CA2387371A1 - Helical cytokine zalpha33 - Google Patents

Abstract

Novel cytokine polypeptides, materials and methods for making them, and meth od of use are disclosed. The polypeptides comprise at least nine contiguous ami no acid residues of SEQ ID NO:2 or SEQ ID NO:4, and may be prepared as polypeptide fusions comprise heterologous sequences, such as affinity tags. The polypeptides and polynucleotides encoding them may be used within a variety of therapeutic, diagnostic, and research applications.

Description

Description BACKGROUND OF THE INVENTION
Cytokines are polypeptide hormones that are produced by a cell and affect the growth or metabolism of that cell or another cell. In multicellular animals, cytokines control cell growth, migration, differentiation, and maturation.
Cytokines play a role in both normal development and pathogenesis, including the development of solid tumors.
Cytokines are physicochemically diverse, ranging in size from 5 kDa (TGF-a) to 140 kDa (Mullerian-inhibiting substance). They include single polypeptide chains, as well as disulfide-linked homodimers and heterodimers.
Cytokines influence cellular events by binding to cell-surface receptors.
Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes such as cell division, protease production, cell migration, expression of cell surface proteins, and production of additional growth factors.

2 0 Cell differentiation and maturation are also under control of cytokines.
For example, the hematopoietic factors erythropoietin, thrombopoietin, and G-CSF
stimulate the production of erythrocytes, platelets, and neutrophils, respectively, from precursor cells in the bone marrow. Development of mature cells from pluripotent progenitors may require the presence of a plurality of factors.
2 5 The role of cytokines in controlling cellular processes makes them likely candidates and targets for therapeutic intervention; indeed, a number of cytokines have been approved for clinical use. Interferon-alpha (IFN-a), for example, is used in the treatment of hairy cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, condylomata acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal and 3 0 Puri, "Common and Uncommon Features of Cytokines and Cytokine Receptors:
An Overview", in Aggarwal and Puri, eds., Human Cytokines: Their Role in Disease and Therapy, Blackwell Science, Cambridge, MA, 1995, 3-24). Platelet-derived growth factor (PDGF) has been approved in the United States and other countries for the treatment of dermal ulcers in diabetic patients. The hematopoietic cytokine 35 erythropoietin has been developed for the treatment of anemias (e.g., EP
613,683). G-CSF, GM-CSF, IFN-(3, IFN-y, and IL,-2 have also been approved for use in humans (Aggarwal and ~uri, ibid.). Experimental evidence supports additional-therapeutic uses of cytokines and their inhibitors. Inhibition of PDGF receptor activity has been shown to reduce intimal hyperplasia in injured baboon arteries (Giese et al., Restenosis Summit VIII, Poster Session #23, 1996; U.S. Patent No. 5,620,687). Vascular endothelial growth factors (VEGFs) have been shown to promote the growth of blood vessels in ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and have been proposed for use as wound-healing agents, for treatment of periodontal disease, for promoting endothelialization in vascular graft surgery, and for promoting collateral circulation following myocardial infarction (WIPO Publication No. WO 95/24473;
U.S.
Patent No. 5,219,739). A soluble VEGF receptor (soluble flt-1) has been found to block binding of VEGF to cell-surface receptors and to inhibit the growth of vascular tissue in vitro (Biotechnology News 16(17):5-6, 1996). Experimental evidence suggests that inhibition of angiogenesis may be used to block tumor development (Biotechnology News, Nov. 13, 1997) and that angiogenesis is an early indicator of cervical cancer (Br. J. Cancer 76:1410-1415, 1997). More recently, thrombopoietin has been shown to stimulate the production of platelets in vivo (Kaushansky et al., Nature 369:568-571, 1994) and has been the subject of several clinical trials (reviewed by von dem Borne et al., Bailliere's Clin. Haematol. 11:427-445, 1998).
In view of the proven clinical utility of cytokines, there is a need in the art for additional such molecules for use as both therapeutic agents and research tools 2 0 and reagents. Cytokines are used in the laboratory to study developmental processes, and in laboratory and industry settings as components of cell culture media.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide novel polypeptides, 2 5 polynucleotides encoding them, and methods of making them.
It is another object of the invention to provide compositions and methods for modulating the proliferation, differentiation, migration, and metabolism of responsive cell types and for regulating tissue development.
Within one aspect of the invention there is provided an isolated 3 0 polypeptide comprising at least nine contiguous amino acid residues of SEQ
ID N0:2 or SEQ ID N0:4. Within one embodiment, the polypeptide has from 15 to 1500 amino acid residues. Within another embodiment, the polypeptide comprises at least nine contiguous amino acid residues of SEQ ID N0:2 or SEQ ID N0:4 operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group 3 5 consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ m N0:7. Within further embodiments, the polypeptide comprises at least 30 contiguous residues of SEQ
ID

N0:2 or SEQ >D N0:4. Within other embodiments, the polypeptide comprises residues 41-55 of SEQ >D N0:2, residues 56-77 of SEQ >D N0:2, residues 78-92 of SEQ >D
N0:2, residues 78-92 of SEQ >D N0:4, residues 93-110 of SEQ )D N0:2, residues 125 of SEQ )D N0:2, residues 111-125 of SEQ >D N0:4, residues 126-148 of SEQ
>D
N0:2, residues 126-148 of SEQ >D N0:4, or residues 149-163 of SEQ >D N0:2.
Within additional embodiments, the polypeptide comprises residues 41-163 of SEQ >D
N0:2, SEQ >D N0:4, or SEQ >D NO:S; residues 34-163 of SEQ >D N0:2, SEQ >D
N0:4, or SEQ >D NO:S; residues 34-178 of SEQ >D N0:2, SEQ >D N0:4, or SEQ >D
NO:S; or residues 18-178 of SEQ >D NO: 2, SEQ 1D N0:4, or SEQ >D NO:S.
Within a second aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter, a DNA segment encoding a polypeptide as disclosed above, and a transcription terminator. Within one embodiment, the DNA segment comprises nucleotides 52 to 534 of SEQ >D N0:6. Within another embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment.
Within a third aspect, the invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses the DNA segment. The cell can be used within a method of making a polypeptide, the method comprising culturing the cell under conditions whereby the DNA segment is 2 0 expressed and the polypeptide is produced, and recovering the polypeptide.
Within one embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, and the polypeptide is secreted by the cell and recovered from a medium in which the cell is cultured.
Within a further aspect of the invention there is provided a polypeptide 2 5 produced by the method disclosed above.
Within another aspect, the invention provides an antibody that specifically binds to the polypeptide disclosed above.
Within an additional aspect, the invention provides a method of detecting, in a test sample, the presence of an antagonist of zalpha33 activity. The 3 0 method comprises the steps of (a) culturing a cell that is responsive to zalpha33; (b) exposing the cell to a zalpha33 polypeptide in the presence and absence of a test sample; (c) comparing levels of response to the zalpha33 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and (d) determining from the comparison the presence of an antagonist of zalpha33 activity in 3 5 the test sample.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID N0:2. The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored.
These residues are indicated in the figure by lower case letters.
Fig. 2 shows an alignment of representative human (SEQ >D N0:2) and mouse (SEQ ID N0:4) zalpha33 polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:
The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol.
198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-2 0 Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985) (SEQ ID N0:7), substance P, Flag'''M peptide (Hopp et al., Biotechnology 6:1204-1210, 1988), streptavidin binding peptide, maltose binding protein (Guan et al., Gene 67:21-30, 1987), cellulose binding protein, thioredoxin, ubiquitin, T7 polymerase, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags and other reagents are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ;
New England Biolabs, Beverly, MA; Eastman Kodak, New Haven, CT).
The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation 3 0 arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
The terms "amino-terminal" and "carboxyl-terminal" are used herein to 3 5 denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
A "complement" of a polynucleotide molecule is a polynucleotide 5 molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
The term "corresponding to", when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when 1 o the sequences are optimally aligned.
The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins 2 0 of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of 2 5 other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' 3 0 untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and 3 5 animal tissue. The isolated polypeptide or protein may be substantially free of other polypeptides or proteins, particularly those of animal origin. An isolated polypeptide or protein may be provided in a highly purified form, i.e. greater than 95% pure or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide or protein in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
"Operably linked" means that two or more entities are joined together such that they function in concert for their intended purposes. When referring to DNA
segments, the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segments) to the terminator. When referring to polypeptides, "operably linked" includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired functions) of the sequences are retained.
The term "ortholog" denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides 2 0 ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length 2 5 and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 3 0 amino acid residues are commonly referred to as "peptides".
The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
3 5 A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone structures;
substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
A "secretory signal sequence" is a DNA sequence that encodes a polypeptide (a °'secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
A "segment" is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.
Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ~10%.
The present invention provides novel cytokine polypeptides and 2 0 proteins. This novel cytokine, termed "zalpha33", was identified by the presence of polypeptide and polynucleotide features characteristic of four-helix-bundle cytokines (e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin, and growth hormone).
Analysis of the amino acid sequence shown in SEQ m N0:2 indicates the presence of four amphipathic, alpha-helical regions. These regions include at least amino acid 2 5 residues 41 through 55 (helix A), 78 through 92 (helix B), 111 through 125 (helix C), and 149 through 163 (helix D). Within these helical regions, residues that are expected to lie within the core of the four-helix bundle occur at positions 41, 44, 45, 48, 51, 52, 55, 78, 81, 82, 85, 88, 89, 92, 111, 114, 115, 118, 121, 122, 125, 149, 152, 153, 156, 159, 160, and 163 of SEQ ID N0:2. Residues 42, 43, 46, 47, 49, 50, 53, 54, 79, 80, 83, 3 0 84, 86, 87, 90, 91, 112, 113, 116, 117, 119, 120, 123, 124, 150, 151, 154, 155, 157, 158, 161, and 162 are expected to lie on the exposed surface of the bundle.
Inter-helix loops comprise approximately residues 56 through 77 (loop A-B), 93 through 110 (loop B-C), and 126 through 148 (loop C-D). The human zalpha33 cDNA (SEQ ID NO:1 ) encodes a polypeptide of 178 amino acid residues. While not wishing to be bound by 3 5 theory, this sequence is predicted to include a secretory peptide of 17 residues.
Cleavage after residue 17 will result in a mature polypeptide (residues 18-178 of SEQ
ID N0:2) having a calculated molecular weight (exclusive of glycosylation) of 18,655 Da. Those skilled in the art will recognize, however, that some cytokines (e.g., endothelial cell growth factor, basic FGF, and IL,-1 (3) do not comprise conventional secretory peptides and are secreted by a mechanism that is not understood. The cDNA
also includes a clear polyadenylation signal, as well as two message instability motifs (ATTTA) in the 3'-untranslated region beginnning at nucleotides 679 and 753.
These message instability motifs are characteristic of cytokine genes (see, e.g., Shaw and Kamen, Cell 46:659-667, 1986).
Those skilled in the art will recognize that predicted domain boundaries are somewhat imprecise and may vary by up to ~ 5 amino acid residues.
1 o Polypeptides of the present invention comprise at least 6, at least 9, or at least 15 contiguous amino acid residues of SEQ >D N0:2. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ >D N0:2, up to the entire predicted mature polypeptide (residues 18 to 178 of SEQ )D N0:2) or the primary translation product (residues 1 to 178 of SEQ >D
N0:2). As disclosed in more detail below, these polypeptides can further comprise additional, non-zalpha33, polypeptide sequence(s). Corresponding mouse zalpha33 polypeptides (see SEQ m N0:4) are also provided by the invention.
Within the polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ )D N0:2 or SEQ m 2 o N0:4. An "epitope" is a region of a protein to which an antibody can bind.
See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984.
Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic 2 5 part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666, 1983.
Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells or 3 0 tissue samples. Antibodies to linear epitopes are also useful for detecting fragments of zalpha33, such as might occur in body fluids or cell culture media.
Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zalpha33 protein. Antigenic, epitope-bearing polypeptides contain a sequence of at 3 5 least six, often at least nine, commonly from 15 to about 30 contiguous amino acid residues of a zalpha33 protein (e.g., SEQ >D N0:2). Polypeptides comprising a larger portion of a zalpha33 protein, i.e. from 30 to 50 residues up to the entire sequence, are included. It is preferred that the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided. Such regions include the interdomain loops of zalpha33 and fragments thereof, particular loop C-D, which is markedly hydrophilic (see Fig. 1 ).
Exemplary polypeptides in this regard include those comprising residues 136-141, 123-128, 119-124, 135-140, or 147-152 of SEQ ID N0:2.
Of particular interest within the present invention are polypeptides that comprise the entire four-helix bundle of a zalpha33 polypeptide (e.g., residues 41-163 of SEQ ID N0:2). Such polypeptides may further comprise all or part of one or both of the native zalpha33 amino-terminal (residues 18-40 of SEQ ID N0:2) and carboxyl-terminal (residues 164-178 of SEQ ID N0:2) regions, as well as non-zalpha33 amino acid residues or polypeptide sequences as disclosed in more detail below.
Polypeptides of the present invention can be prepared with one or more amino acid substitutions, deletions or additions as compared to SEQ ID N0:2.
These changes will usually be of a minor nature, that is conservative amino acid substitutions and other changes that do not significantly affect the folding or activity of the protein or polypeptide, and include amino- or carboxyl-terminal extensions, such as an amino terminal methionine residue, an amino or carboxyl-terminal cysteine residue to 2 0 facilitate subsequent linking to maleimide-activated keyhole limpet hemocyanin, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as disclosed above. Two or more affinity tags may be used in combination. Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zalpha33 polypeptide 2 5 and the affinity tag. Exemplary cleavage sites include thrombin cleavage sites and factor Xa cleavage sites.
The present invention further provides a variety of other polypeptide fusions. For example, a zalpha33 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
Suitable 3 0 dimerizing proteins in this regard include immunoglobulin constant region domains.
Immunoglobulin-zalpha33 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zalpha33 analogs. In addition, a zalpha33 polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a mufti-functional molecule. One or more helices of a zalpha33 polypeptide 3 5 can be joined to another cytokine to enhance or otherwise modify its biological properties. Auxiliary domains can be fused to zalpha33 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a zalpha33 polypeptide or protein can be targeted to a predetermined cell type by fusing a zalpha33 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zalpha33 polypeptide can be fused to two or more moieties, such as an 5 affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
Polypeptide fusions of the present invention will generally contain not more than about 1,500 amino acid residues, often not more than about 1,200 residues, 10 frequently not more than about 1,000 residues, and will in many cases be considerably smaller. For example, a zalpha33 polypeptide of 161 residues (residues 18-178 of SEQ
ID N0:2) can be fused to E. coli ~3-galactosidase (1,021 residues; see Casadaban et al., J. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xa cleavage site to yield a polypeptide of 1,196 residues. In a second example, residues 18-178 of SEQ >D N0:2 can be fused to maltose binding protein (approximately residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.
As disclosed above, the polypeptides of the present invention comprise at least 6 contiguous residues of SEQ >D N0:2 or SEQ >D N0:4. These polypeptides may further comprise additional residues as shown in SEQ >D N0:2, a variant of SEQ
2 0 >D N0:2, or another protein as disclosed herein. "Variants of SEQ >D N0:2"
includes polypeptides that are at least 90% or at least 95% identical to the corresponding region of SEQ >D N0:2. Percent sequence identity is determined by conventional methods.
See, for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992. Briefly, two amino acid 2 5 sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:
3 0 Total number of identical matches x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two 3 5 sequences]

> ~t ~. N M

E-' v~N N O

C/~ ~ ,-,M N N

p., ~ ~ ~ ~ M N

LZ. ~p ~ N N .-,M

i i i i M ~ O ~ M N N

r~ d'N N O

""' '~tN M -.O M N ~ M ~~M

H

ppM M ~ N .-~N N N N M

i N d' ~ N M M N O N N M M

w ~ N O M M .~ N M ~ O ~ M N N

V7 N N O M N --.O M ~ O '-'N '-'N

M ~ M M ~ .-M -~N M .~ ~ N N i , I

L \p M O N .~M ~ ~ M M .-~ M M

~ ~ O ~

z ~ ~ M O O O ~ n; ~";p N M N ,.,O V N M

O N M ,_,O N O M N N ~ M N ~ ~ M N M

Q ~ i N N O i ; O N ,~.-..,~N .~-. M N

~ . O O

x z r~ v a w ~ x ~ a x ~ w a.~ H 3 ~ >

The level of identity between amino acid sequences can be determined using the "FASTA" similarity search algorithm disclosed by Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988) and by Pearson (Meth. Enzymol.
183:63, 1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ >D N0:2) and a test sequence that have either the highest density of identities (if the ktup variable is 1 ) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff' value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), which allows for amino acid insertions and deletions. Preferred parameters for FASTA
analysis are: ktup=1, gap opening penalty=10, gap extension penalty=1, and 2 0 substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA
program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, 1990 (ibid.).
FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, 2 5 the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
The present invention includes polypeptides having one or more conservative amino acid changes as compared with the amino acid sequence of SEQ ID
N0:2. The BLOSUM62 matrix (Table 1) is an amino acid substitution matrix derived 3 0 from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, ibid.). Thus, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the term "conservative amino acid 3 5 substitution" refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least one 1 (e.g., l, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
The proteins of the present invention can also comprise non-naturally occuring amino acid residues. Non-naturally occuring amino acids include, without limitation, traps-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, traps-4 hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4 azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-809, 1993; Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-10149, 1993; Turcatti et al., J. Biol. Chem.
271:19991-19998, 1996; and Koide et al., Biochem. 33:7470-7476, 1994. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
Amino acid sequence changes are made in zalpha33 polypeptides so as to minimize disruption of higher order structure essential to biological activity. For 2 0 example, changes in amino acid residues will be made so as not to disrupt the four helix bundle characteristic of the protein family. The effects of amino acid sequence changes can be predicted by computer modeling using available software (e.g., the Insight II~ viewer and homology modeling tools; MSI, San Diego, CA) or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nature 369:455-461, 1994 and Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). A hydrophilicity profile of SEQ ID
N0:2 is shown in Fig. 1. Those skilled in the art will recognize that this hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a zalpha33 polypeptide, so as not to disrupt the overall profile. Residues within the core of the four-helix bundle can be replaced with a hydrophobic residue selected from the 3 0 group consisting of Leu, Ile, Val, Met, Phe, Trp, Gly, and Ala as shown in SEQ ID
NO:S. Cysteine residues at positions 99 and 103 of SEQ ID N0:2 and SEQ ID N0:4 and the residues predicted to be on the exposed surface of the four-helix bundle will be relatively intolerant of substitution. Other candidate amino acid substitutions within human zalpha33 are suggested by alignment of the human (SEQ >D N0:2) and mouse 3 5 (SEQ >D N0:4) sequences as shown in Fig. 2, which sequences are approximately 95%
identical overall.

One skilled in the art may employ many well known techniques, independently or in combination, to analyze and compare the structural features that affect folding of a variant protein or polypeptide to a standard molecule to determine whether such modifications would be significant. One well known and accepted method for measuring folding is circular dichroism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule are routine in the art (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known and accepted method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are other known methods for analyzing folding and structurally similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).
Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule.
Multiple amino acid substitutions can be made and tested using known 2 0 methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA
86:2152-2156, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the 2 5 spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;
Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA
7:127, 1988).
3 0 Variants of the disclosed zalpha33 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751, 1994. Briefly, variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced 3 5 point mutations. This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution"
of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
In many cases, the structure of the final polypeptide product will result 5 from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a zalpha33 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide. For example, expressing the complete zalpha33 sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a 10 prokaryotic host would not be expected to be cleaved. Differential processing of individual chains may result in heterogeneity of expressed polypeptides.
The human and mouse zalpha33 polypeptide sequences each contain 3 cysteine residues, at positions 34, 99, and 103. Structural predictions indicate that Cys99 and Cys103 may form an intrachain disulfide bond, and that Cys34 may be free 15 to form an interchain disulfide bond, resulting in dimerization. Actual conformation will depend in part upon the cell in which in the polypeptide is expressed.
The polypeptides of the present invention thus include those comprising these cysteine residues, such as polypeptides comprising residues 34-163 or 34-178 of SEQ >D
N0:2 or SEQ >D N0:4.
2 0 Zalpha33 proteins of the present invention are characterized by their activity, that is, modulation of the proliferation, differentiation, migration, adhesion, or metabolism of responsive cell types. Responsive cell types include epithelial cells and the cell lines U-373 MG (human brain glioblastoma) and 3A-Sub E (SV40-transformed human placenta). Biological activity of zalpha33 proteins is assayed using in vitro or in vivo assays designed to detect cell proliferation, differentiation, migration or adhesion;
or changes in cellular metabolism (e.g., production of other growth factors or other macromolecules). Many suitable assays are known in the art, and representative assays are disclosed herein. Assays using cultured cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions.
3 0 However, in view of the complexity of developmental processes (e.g., angiogenesis, wound healing), in vivo assays will generally be employed to confirm and further characterize biological activity. Certain in vitro models, such as the three-dimensional collagen gel matrix model of Pepper et al. (Biochem. Biophys. Res. Comm.
189:824-831, 1992), are sufficiently complex to assay histological effects. Assays can be performed using exogenously produced proteins, or may be carried out in vivo or in vitro using cells expressing the polypeptide(s) of interest. Assays can be conducted using zalpha33 proteins alone or in combination with other growth factors, such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative assays are disclosed below.
Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zalpha33 variant polypeptides. Assays that can be scaled up for high throughput include mitogenesis assays, which can be run in a 96-well format. Mutagenized DNA
molecules that encode active zalpha33 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptide fragments or variants of SEQ )D N0:2 or SEQ
>D
N0:4 that retain the activity of wild-type zalpha33.
The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zalpha33 polypeptides disclosed above.
A
representative DNA sequence encoding the amino acid sequence of SEQ >D N0:2 is shown in SEQ ID NO:1, and a representative DNA sequence encoding the amino acid sequence of SEQ >D N0:4 is shown in SEQ >D N0:3. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable 2 0 sequence variation is possible among these polynucleotide molecules. SEQ
ID N0:6 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha33 polypeptide of SEQ m NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID N0:6 also provides all RNA sequences encoding SEQ
ID N0:2 by substituting U for T. Thus, zalpha33 polypeptide-encoding polynucleotides comprising nucleotides 1-534 or nucleotides 52-534 of SEQ m N0:6, and their RNA
equivalents are contemplated by the present invention, as are segments of SEQ
>D N0:6 encoding other zalpha33 polypeptides disclosed herein. Table 2 sets forth the one-letter codes used within SEQ ID N0:6 to denote degenerate nucleotide positions.
"Resolutions" are the nucleotides denoted by a code letter. "Complement"
indicates the 3 0 code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G
being complementary to C.

Nucleotide Resolutions Complement Resolutions A A T T

C C G G

G G C C

T T A A

R A~G Y C~T

Y C~T R A~G

M A~C K G~T

K G~T M A~C

S C~G S C~G

W A~T W A~T

H A~C~T D A~G~T

B C~G~T V A~C~G

V A~C~G B C~G~T

D A~G~T H A~C~T

N A~C~G~T N A~C~G~T

The degenerate codons used in SEQ >D N0:6, encompassing all possible codons for a given amino acid, are set forth in Table 3, below.

Amino One-Letter Degenerate Acid Code Codons Codon Cys C TGC TGT TGY

Ser S AGC AGT TCA TCC TCG TCT WSN

Thr T ACA ACC ACG ACT CAN

Pro P CCA CCC CCG CCT CCN

Ala A GCA GCC GCG GCT GCN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT AAY

Asp D GAC GAT GAY

Glu E GAA GAG GAR

Gln Q CAA CAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG

Ile I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

V al V GTA GTC GTG GTT GTN

Phe F TTC TTT TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter . TAA TAG TGA TRR

Asn~AspB RAY

Glu~GlnZ SAR

Any X NNN

Gap One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ m NO: 2. Variant sequences can be readily tested for functionality as described herein.
One of ordinary skill in the art will also appreciate that different species can exhibit preferential codon usage. See, in general, Grantham et al., Nuc.
Acids Res.
8:1893-912, 1980; Haas et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.
14:3075-87, 1986; and Ikemura, J. Mol. Biol. 158:573-97, 1982. Introduction of preferred codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID N0:6 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein.
Within certain embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ 117 NO:1 or SEQ
m N0:3, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence 2 0 hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well 2 5 known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zalpha33 RNA. Suitable tissues include heart, liver, pancreas, testis, ovary, and thyroid. Zalpha33 transcripts have also been detected in B-cells and in many tumor cells. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCI gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
3 0 Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc.
Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly(A)+ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zalpha33 polypeptides are then identified and isolated by, for example, hybridization or PCR.
3 5 Full-length clones encoding zalpha33 can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are often preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with 5 antibodies to zalpha33, receptor fragments, or other specific binding partners.
Zalpha33 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zalpha33 gene. Promoter elements from a zalpha33 gene could thus be used to direct the expression of heterologous genes in, for example, transgenic animals or patients treated with gene 1 o therapy. Cloning of 5' flanking sequences also facilitates production of zalpha33 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670.
Briefly, expression of an endogenous zalpha33 gene in a cell is altered by introducing into the zalpha33 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a 15 zalpha33 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zalpha33 locus, whereby the sequences within the construct become operably linked with the endogenous zalpha33 coding sequence.
In this way, an endogenous zalpha33 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated 2 0 expression.
A human zalpha33 gene sequence is shown in SEQ ID NO:10. Within SEQ >D NO:10, exons are at nucleotides 1001-1319, 8914-9072, 10,986-11,090, and 20,144-20,597. Nucleotides 1-1000 of SEQ ID N0:2 are believed to contain promoter and regulatory elements.
2 5 Those skilled in the art will recognize that the sequences disclosed in SEQ >D NOS:1 and 2 represent a single allele of human zalpha33, and the sequences disclosed in SEQ m NOS:3 and 4 represent a single allele of mouse zalpha33.
Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
3 0 The present invention further provides counterpart polypeptides and polynucleotides from other species ("orthologs"). Of particular interest are zalpha33 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zalpha33 can be cloned using information and compositions provided by the present invention in 3 5 combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zalpha33 as disclosed above. A library is then prepared from mRNA of a positive tissue or cell line.

A zalpha33-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zalpha33 sequence disclosed herein.
Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zalpha33 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
For any zalpha33 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 3 and 4, above.
Moreover, those of skill in the art can use standard software to devise zalpha33 variants based upon the nucleotide and amino acid sequences described herein. The present invention thus provides a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ >D NO:1, SEQ >D N0:2, SEQ >D
N0:3, SEQ )D N0:4, SEQ >D NO:S, SEQ 1D N0:6, and portions thereof. Suitable forms of computer-readable media include magnetic media and optically-readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIPTM
2 0 disk. Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
The zalpha33 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides can be produced 2 5 according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide. As used herein, "cells into which have been introduced an expression vector" include both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA. Suitable host cells are those cell types that can be 3 0 transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and 3 5 Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zalpha33 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome.
Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are 1 o described in the literature and are available through commercial suppliers.
To direct a zalpha33 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zalpha33, or may be derived from another secreted protein (e.g., t-PA; see, U.S. Patent No. 5,641,655) or synthesized de novo. The secretory signal sequence is operably linked to the zalpha33 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly sythesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal 2 0 sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No.
5,143,830).
Cultured mammalian cells can be used as hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;
Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for 3 0 example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S.
Patent No.
4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S.
Patent No.
4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL
1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol.
3 5 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61;
or CHO DG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines.
Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA, USA.
In general, strong transcription promoters are will be used, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter. Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited with the American Type Culture Collection, Manassas, VA, USA under accession numbers 98669 and 98668, respectively, and derivatives thereof.
Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." An exemplary selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification."
Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. An exemplary amplifiable 2 o selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used.
The adenovirus system (disclosed in more detail below) can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under 2 5 conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. In an alternative 3 0 method, adenovirus vector-infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Garnier et al., Cytotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the 3 5 expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins can also be effectively obtained.

Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) according to methods known in the art. Within one method, recombinant baculovirus is produced through the use of a transposon-based system described by Luckow et al. (J.
Virol.
67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (Bac-to-BacT"" kit; Life Technologies, Rockville, MD).
The transfer vector (e.g., pFastBaclT""; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." See, Hill-Perkins and Possee, J. Gen.
Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556, 1994;
and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above. Using techniques known in the art, a transfer vector containing a zalpha33-encoding sequence is transformed into E. coli host cells, and the cells are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. Recombinant virus that expresses zalpha33 protein is subsequently produced. Recombinant viral stocks are made by methods commonly 2 0 used the art.
For protein production, the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveT"" cells; Invitrogen, Carlsbad, CA).
See, for example, U.S. Patent No. 5,300,435. Serum-free media are used to grow and 2 5 maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers. The cells are grown up from an inoculation density of approximately 2-5 x 105 cells to a density of 1-2 x 106 cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally known in the art.
3 0 Other higher eukaryotic cells can also be used as hosts, including plant cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci.
(Bangalore) 11:47-58, 1987.
Fungal cells, including yeast cells, can also be used within the present 3 5 invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S.
cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311;
Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008;
Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
Transformed cells are selected by phenotype determined by the selectable marker, 5 commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). An exemplary vector system for use in Saccharomyces cerevisiae is the POTI vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those from glycolytic 10 enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S.
Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154;
5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago 15 maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen.
Microbiol.
132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Raymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum 20 are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Patents No.
5,716,808, 5,736,383, 5,854,039, and 5,888,768.
Prokaryotic host cells, including strains of the bacteria Escherichia coli, 2 5 Bacillus and other genera are also useful host cells within the present invention.
Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a zalpha33 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space 3 0 by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the 3 5 latter case; the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
The polypeptides and proteins of the present invention can be purified to >_80% purity, to ?90% purity, to >_95% purity, or to a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
The desired degree of purification will depend upon the intended use of the polypeptide or protein.
2 0 Expressed recombinant zalpha33 proteins (including chimeric polypeptides and multimeric proteins) are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromato~raphy: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, 2 5 Springer-Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bioll'echnol. 6: 1321-1325, 1988.
Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid.
3 0 Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
Zalpha33 polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, 3 5 for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984;
Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis:

A Practical Approach, 1RL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.
PEGylation is one method commonly used that has been demonstrated to increase plasma half life, increase solubility, and decrease antigenicity and immunogenicity of proteins (Nucci et al., Advanced Drug Delivery Reviews 6:133-155, 1991 and Lu et al., Int. J. Peptide Protein Res. 43:127-138, 1994). Several procedures for creating and purifying pegylated proteins are known in the art. See, for example, Abuchowski et al., J. Biol. Chem. 252:3582-3586, 1977 and Becauchamp et al., Anal.
Biochem. 131:25-33, 1983. Particular acid amino acid residues (e.g. glutamic and aspartic acids) and amino acids at the carboxyl-terminus of a protein are amenable to PEGylation (Zalipsky, Bioconjugate Chem. 6:150-165, 1995).
Using methods known in the art, zalpha33 proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated;
and may or may not include an initial methionine amino acid residue.
Target cells for use in zalpha33 activity assays include, without limitation, vascular cells (especially endothelial cells and smooth muscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts (including human dermal fibroblasts and lung fibroblasts), fetal lung cells, articular synoviocytes, 2 0 pericytes, chondrocytes, osteoblasts, and prostate epithelial cells.
Endothelial cells and hematopoietic cells are derived from a common ancestral cell, the hemangioblast (Choi et al., Development 125:725-732, 1998).
Activity of zalpha33 proteins can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate 2 5 animal model. Assays measuring cell proliferation or differentiation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (as disclosed by, e.g., Raines and Ross, Methods Enzymol. 109:749-773, 1985; Wahl et al., Mol. Cell Biol.
3 0 8:5016-5025, 1988; and Cook et al., Analytical Biochem. 179:1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J.
Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;
Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res.
48:4827-3 5 4833, 1988). Differentiation can be assayed using suitable precursor cells that can be induced to differentiate into a more mature phenotype. Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989;
all incorporated herein by reference).
Zalpha33 activity may also be detected using assays designed to measure zalpha33-induced production of one or more additional growth factors or other macromolecules. Such assays include those for determining the presence of hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFa), interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF), l0 angiogenin, and other macromolecules produced by the liver. Suitable assays include mitogenesis assays using target cells responsive to the macromolecule of interest, receptor-binding assays, competition binding assays, immunological assays (e.g., ELISA), and other formats known in the art. Metalloprotease secretion is measured from treated primary human dermal fibroblasts, synoviocytes and chondrocytes.
The relative levels of collagenase, gelatinise and stromalysin produced in response to culturing in the presence of a zalpha33 protein is measured using zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106, 1990). Procollagen/collagen synthesis by dermal fibroblasts and chondrocytes in response to a test protein is measured using 3H-proline incorporation into nascent secreted collagen. 3H-labeled 2 0 collagen is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG) secretion from dermal fibroblasts and chondrocytes is measured using a 1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim. Biophys.
Acta 883:173-177, 1986). Collagen and GAG assays are also carried out in the presence of 2 5 IL.-1 (3 or TGF-(3 to examine the ability of zalpha33 protein to modify the established responses to these cytokines.
Monocyte activation assays are carried out (1) to look for the ability of zalpha33 proteins to further stimulate monocyte activation, and (2) to examine the ability of zalpha33 proteins to modulate attachment-induced or endotoxin-induced 3 0 monocyte activation (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987).
IL-1 (3 and TNFa levels produced in response to activation are measured by ELISA
(Biosource, Inc. Camarillo, CA). Monocyte/macrophage cells, by virtue of CD14 (LPS
receptor), are exquisitely sensitive to endotoxin, and proteins with moderate levels of endotoxin-like activity will activate these cells.
3 5 Hematopoietic activity of zalpha33 proteins can be assayed on various hematopoietic cells in culture. Suitable assays include primary bone marrow colony assays and later stage lineage-restricted colony assays, which are known in the art (e.g., Holly et al., WIPO Publication WO 95/21920). Marrow cells plated on a suitable semi-solid medium (e.g., 50% methylcellulose containing 15% fetal bovine serum, 10%
bovine serum albumin, and 0.6% PSN antibiotic mix) are incubated in the presence of test polypeptide, then examined microscopically for colony formation. Known hematopoietic factors are used as controls. Mitogenic activity of zalpha33 polypeptides on hematopoietic cell lines can be measured as disclosed above.
Cell migration is assayed essentially as disclosed by Kahler et al.
(Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). A
protein is considered to be chemotactic if it induces migration of cells from an area of low protein concentration to an area of high protein concentration. A typical assay is performed using modified Boyden chambers with a polystryrene membrane separating the two chambers (e.g., Transwell~; Corning Costar Corp.). The test sample, diluted in medium containing 1 % BSA, is added to the lower chamber of a 24-well plate containing Transwells. Cells are then placed on the Transwell insert that has been pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37°C. Non-migrating cells are wiped off the top of the Transwell membrane, and cells attached to the lower face of the membrane are fixed and stained with 0.1 %
crystal violet. Stained cells are then extracted with 10% acetic acid and absorbance is measured at 600 nm. Migration is then calculated from a standard calibration curve.
2 0 Cell migration can also be measured using the matrigel method of Grant et al.
("Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997).
Growth factor effects of zalpha33 proteins can also be assayed in an aortic ring outgrowth assay (Nicosia and Ottinetti, Laboratory Investigation 63:115, 1990; Villaschi and Nicosia, Am. J. Pathology 143:181-190, 1993).
Cell adhesion activity is assayed essentially as disclosed by LaFleur et al.
(J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are coated with the test protein, non-specific sites are blocked with BSA, and cells (such as smooth muscle 3 0 cells, leukocytes, or endothelial cells) are plated at a density of approximately 104 - 105 cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then non adherent cells are removed by gentle washing. Adhered cells are quantitated by conventional methods (e.g., by staining with crystal violet, lysing the cells, and determining the optical density of the lysate). Control wells are coated with a known 3 5 adhesive protein, such as fibronectin or vitronectin.
The activity of zalpha33 proteins can be measured with a silicon-based biosensor microphysiometer that measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses. An exemplary such device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, 5 regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257:1906-1912, 1992; Pitchford et al., Meth.
Enzymol. 228:84-108, 1997; Arimilli et al., J. Immunol. Meth. 212:49-59, 1998;
and Van Liefde et al., Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By 10 measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zalpha33 proteins, their agonists, and antagonists. The microphysiometer can be used to measure responses of a zalpha33-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zalpha33 polypeptide. Zalpha33-responsive 15 eukaryotic cells comprise cells into which a receptor for zalpha33 has been transfected creating a cell that is responsive to zalpha33, as well as cells naturally responsive to zalpha33 such as cells derived from vascular tissue. Differences, measured by a change in extracellular acidification, in the response of cells exposed to zalpha33 polypeptide relative to a control not exposed to zalpha33, are a direct measurement of zalpha33-2 0 modulated cellular responses. Moreover, such zalpha33-modulated responses can be assayed under a variety of stimuli. The present invention thus provides methods of identifying agonists and antagonists of zalpha33 proteins, comprising providing cells responsive to a zalpha33 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test 2 5 compound, and detecting a change in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change in extracellular acidification rate. Culturing a third portion of the cells in the presence of a zalpha33 protein and the absence of a test compound provides a positive control for the zalpha33-responsive cells and a control to 3 0 compare the agonist activity of a test compound with that of the zalpha33 polypeptide.
Antagonists of zalpha33 can be identified by exposing the cells to zalpha33 protein in the presence and absence of the test compound, whereby a reduction in zalpha33-stimulated activity is indicative of antagonist activity in the test compound.
Expression of zalpha33 polynucleotides in animals provides models for 3 5 further study of the biological effects of overproduction or inhibition of protein activity in vivo. Zalpha33-encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.
One in vivo approach for assaying proteins of the present invention utilizes viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth.
Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997. The adenovirus system offers several advantages. Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co transfected plasmid. In an exemplary system, the essential E 1 gene is deleted from the viral vector, and the virus will not replicate unless the E1 gene is provided by the host cell (e.g., the human 293 cell line). When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an E1 gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
2 5 An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAF dextran, calcium 3 0 phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chenz. 263:14621-14624, 1988; Wu et al., J. Biol. Chem.
267:963-967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
Transgenic mice, engineered to express a zalpha33 gene, and mice that exhibit a complete absence of zalpha33 gene function, referred to as "knockout mice"
3 5 (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-742, 1993). These mice can be employed to study the zalpha33 gene and the protein encoded thereby in an in vivo system. Transgenic nice are particularly useful for investigating the role of zalpha33 proteins in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or underexpression of a specific factor. See also, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science 277:48-50, 1997. Suitable promoters for transgenic expression include promoters from metallothionein and albumin genes.
Antisense methodology can be used to inhibit zalpha33 gene transcription to examine the effects of such inhibition in vivo.
Polynucleotides that are complementary to a segment of a zalpha33-encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ >D NO:1) are designed to bind to zalpha33-encoding mRNA and to inhibit translation of such mRNA. Such antisense oligonucleotides can also be used to inhibit expression of zalpha33 polypeptide-encoding genes in cell culture.
Most four-helix bundle cytokines as well as other proteins produced by activated lymphocytes play an important biological role in cell differentiation, activation, recruitment and homeostasis of cells throughout the body. Zalpha33 and inhibitors of zalpha33 activity are expected to have a variety of therapeutic applications.
These therapeutic applications include treatment of diseases which require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, and diabetes.
Zalpha33 may 2 0 be important in the regulation of inflammation, and therefore would be useful in treating rheumatoid arthritis, asthma and sepsis. There may be a role of zalpha33 in mediating tumorigenesis, whereby a zalpha33 antagonist would be useful in the treatment of cancer. Zalpha33 may be useful in modulating the immune system, whereby zalpha33 and zalpha33 antagonists may be used for reducing graft rejection, 2 5 preventing graft-vs-host disease, boosting immunity to infectious diseases, treating immunocompromised patients (e.g., HIV+ patients), or in improving vaccines.
Zalpha33 polypeptides can be administered alone or in combination with other vasculogenic or angiogenic agents, including VEGF. When using zalpha33 in combination with an additional agent, the two compounds can be administered 3 0 simultaneously or sequentially as appropriate for the specific condition being treated.
For pharmaceutical use, zalpha33 proteins are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. In general, pharmaceutical formulations will include a zalpha33 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered 35 saline, 5% dextrose in water, or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995. Zalpha33 will usually be used in a concentration of about 10 to 100 ~g/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 ~.g/ml may be used. For topical application, such as for the promotion of wound healing, the protein will be applied in the range of 0.1-10 ~g/cm2 of wound area, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. Dosing is daily or intermittently over the period of treatment.
Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed. In general, a therapeutically effective amount of zalpha33 is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in hematopoietic or immune function, a significant reduction in morbidity, or a significantly increased histological score.
Zalpha33 proteins, agonists, and antagonists are useful for modulating the expansion, proliferation, activation, differentiation, migration, or metabolism of responsive cell types, which include both primary cells and cultured cell lines. Of particular interest in this regard are hematopoietic cells (including stem cells and 2 0 mature myeloid and lymphoid cells), endothelial cells, smooth muscle cells, fibroblasts, and hepatocytes. Zalpha33 polypeptides are added to tissue culture media for these cell types at a concentration of about 10 pg/ml to about 100 ng/ml. Those skilled in the art will recognize that zalpha33 proteins can be advantageously combined with other growth factors in culture media.
2 5 Within the laboratory research field, zalpha33 proteins can also be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over-or under-production of zalpha33 protein or in the analysis of cell phenotype.
Zalpha33 proteins can also be used to identify inhibitors of their activity.
3 0 Test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of zalpha33 protein. In addition to those assays disclosed above, samples can be tested for inhibition of zalpha33 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zalpha33-dependent cellular responses. For example, zalpha33-responsive cell lines can be 3 5 transfected with a reporter gene construct that is responsive to a zalpha33-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zalpha33-activated serum response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zalpha33 on the target cells as evidenced by a decrease in zalpha33 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zalpha33 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of zalpha33 binding to receptor using zalpha33 tagged with a detectable label (e.g., ~25I, biotin, horseradish peroxidase, FTTC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zalpha33 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody).
In some 2 0 instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics.
Through humanizing antibodies, biological half life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. One skilled in the art can generate humanized antibodies with specific and different constant domains 2 5 (i.e., different Ig subclasses) to facilitate or inhibit various immune functions associated with particular antibody constant domains. Antibodies are defined to be specifically binding if they bind to a zalpha33 polypeptide or protein with an affinity at least 10-fold greater than the binding affinity to control (non-zalpha33) polypeptide or protein. The affinity of a monoclonal antibody can be readily determined by one of ordinary skill in 3 0 the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).
Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982, which is incorporated herein by reference). As would be evident to one of ordinary 3 5 skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of a zalpha33 polypeptide may be increased through the use of an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zalpha33 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a 5 full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
Alternative techniques for generating or selecting antibodies include in 10 vitro exposure of lymphocytes to zalpha33 polypeptides, and selection of antibody display libraries in phage or similar vectors (e.g., through the use of immobilized or labeled zalpha33 polypeptide). Human antibodies can be produced in transgenic, non human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous 15 immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zalpha33 polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), 2 0 Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
Antibodies to zalpha33 may be used for affinity purification of the 2 5 protein, within diagnostic assays for determining circulating levels of the protein; for detecting or quantitating soluble zalpha33 polypeptide as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block protein activity in vitro and in vivo. Antibodies to zalpha33 may 3 0 also be used for tagging cells that express zalpha33; for affinity purification of zalpha33 polypeptides and proteins; in analytical methods employing FACS; for screening expression libraries; and for generating anti-idiotypic antibodies. Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targetting of those compounds to cells expressing receptors for 3 5 zalpha33. For certain applications, including in vitro and in vivo diagnostic uses, it is advantageous to employ labeled antibodies. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies of the present invention may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications(e.g., inhibition of cell proliferation). See, in general, Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.
Polypeptides and proteins of the present invention can be used to identify and isolate receptors. Zalpha33 receptors may be involved in growth regulation in the liver, blood vessel formation, and other developmental processes. For example, zalpha33 proteins and polypeptides can be immobilized on a column, and membrane preparations run over the column (as generally disclosed in Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and polypeptides can also be radiolabeled (Methods Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Academic Press, San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et al., Ann. Rev.
Biochem.
62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and used to tag specific cell-surface proteins. In a similar manner, radiolabeled zalpha33 proteins and polypeptides can be used to clone the cognate receptor in binding assays using cells transfected with an expression cDNA library.
2 o The present invention also provides reagents for use in diagnostic applications. For example, the zalpha33 gene, a probe comprising zalpha33 DNA
or RNA, or a subsequence thereof can be used to determine the presence of mutations at or near the zalpha33 locus. Detectable chromosomal aberrations at the zalpha33 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, 2 5 deletions, restriction site changes, and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.
Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter 3o probes (14-17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, often 15 or more nt, commonly 20-30 nt. Short polynucleotides can be used when a small region of the gene is targetted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more. Probes will generally comprise a polynucleotide linked to a signal-generating moiety such as a 3 5 radionucleotide. In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (c) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ >D NO:1, the complement of SEQ >D NO:1, or an RNA equivalent thereof.
Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.;
Ausubel et. al., ibid.; A.J. Marian, Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g., Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA
probe to a patient RNA sample, after which the reaction product (RNA-RNA
hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion.
Within PCR assays, a patient genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered.
Changes in size or amount of recovered product are indicative of mutations in the patient. Another 2 0 PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-38, 1991 ).
Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et 2 5 al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels that cover the entire human genome are commercially available, such as the Stanford G3 RH Panel and the GeneBridge 4 RH
Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid, PCR-based 3 0 chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest, and the establishment of directly proportional physical distances between newly discovered genes of interest and previously mapped markers. The precise knowledge of a gene's position can be useful for a number of purposes, including: 1 ) determining if a sequence 3 5 is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences.
The polypeptides, nucleic acid and/or antibodies of the present invention may be used in diagnosis or treatment of disorders associated with cell loss or abnormal cell proliferation (including cancer). Labeled zalpha33 polypeptides may be used for imaging tumors or other sites of abnormal cell proliferation.
Inhibitors of zalpha33 activity (zalpha33 antagonists) include anti zalpha33 antibodies and soluble zalpha33 receptors, as well as other peptidic and non 2 0 peptidic agents (including ribozymes). Such antagonists can be used to block the effects of zalpha33 on cells or tissues. Of particular interest is the use of antagonists of zalpha33 activity in cancer therapy. As early detection methods improve it becomes possible to intervene at earlier times in tumor development, making it feasible to use inhibitors of growth factors to block cell proliferation, angiogenesis, and other events 2 5 that lead to tumor development and metastasis. Inhibitors are also expected to be useful in adjunct therapy after surgery to prevent the growth of residual cancer cells.
Inhibitors can also be used in combination with other cancer therapeutic agents.
In addition to antibodies, zalpha33 inhibitors include small molecule inhibitors and inactive receptor-binding fragments of zalpha33 polypeptides.
Inhibitors 3 0 are formulated for pharmaceutical use as generally disclosed above, taking into account the precise chemical and physical nature of the inhibitor and the condition to be treated.
The relevant determinations are within the level of ordinary skill in the formulation art.
Polynucleotides encoding zalpha33 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zalpha33 activity. If a 3 5 mammal has a mutated or absent zalpha33 gene, a zalpha33 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zalpha33 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
Defective viruses, which entirely or almost entirely lack viral genes, are commonly used. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HS V 1 ) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-630, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol. 61:3096-3101, 1987;
Samulski et al., J. Virol. 63:3822-3888, 1989). Within another embodiment, a zalpha33 gene can be introduced in a retroviral vector as described, for example, by Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J.
Virol.
62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty et al., WIPO
Publication WO 95/07358; and Kuo et al., Blood 82:845, 1993. Alternatively, the vector can be introduced by liposome-mediated transfection ("lipofection").
Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987;
2 0 Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages, including molecular targeting of liposomes to specific cells.
Directing transfection to particular cell types is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically 2 5 coupled to other molecules for the purpose of targeting. Peptidic and non-peptidic molecules can be coupled to liposomes chemically. Within another embodiment, cells are removed from the body, a vector is introduced into the cells as a naked DNA
plasmid, and the transformed cells are re-implanted into the body as disclosed above.
Antisense methodology can be used to inhibit zalpha33 gene 3 0 transcription in a patient as generally disclosed above.
Zalpha33 polypeptides and anti-zalpha33 antibodies can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention may be used to identify or treat tissues or organs that 3 5 express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, zalpha33 polypeptides or anti-zalpha33 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues, or organs that express the anti-complementary molecule.
Suitable detectable molecules can be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, 5 inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like. Suitable cytotoxic molecules can be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90. These can be either 10 directly attached to the polypeptide or antibody, or indirectly attached according to known methods, such as through a chelating moiety. Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule may be conjugated with a member of a complementary/anticomplementary pair, where the 15 other member is bound to the polypeptide or antibody portion. For these purposes, biotin/streptavidin is an exemplary complementary/anticomplementary pair.
Polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins may be used for targeted cell or tissue inhibition or ablation, such as in cancer therapy. Of particular interest in this regard are conjugates of a zalpha33 polypeptide 2 0 and a cytotoxin, which can be used to target the cytotoxin to a tumor or other tissue that is undergoing undesired angiogenesis or neovascularization. Target cells (i.e., those displaying the zalpha33 receptor) bind the zalpha33-toxin conjugate, which is then internalized, killing the cell. The effects of receptor-specific cell killing (target ablation) are revealed by changes in whole animal physiology or through histological 2 5 examination. Thus, ligand-dependent, receptor-directed cyotoxicity can be used to enhance understanding of the physiological significance of a protein ligand.
An exemplary toxin is saporin. Mammalian cells have no receptor for saporin, which is non-toxic when it remains extracellular.
In another embodiment, zalpha33-cytokine fusion proteins or 3 0 antibody/fragment-cytokine fusion proteins may be used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers). See, generally, Hornick et al., Blood 89:4437-4447, 1997). In general, cytokines are toxic if administered systemically. The described fusion proteins 3 5 enable targeting of a cytokine to a desired site of action, such as a cell having binding sites for zalpha33, thereby providing an elevated local concentration of cytokine.
Suitable cytokines for this purpose include, for example, interleukin-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Such fusion proteins may be used to cause cytokine-induced killing of tumors and other tissues undergoing angiogenesis or neovascularization.
The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intra-arterially or intraductally, or may be introduced locally at the intended site of action.
The invention is further illustrated by the following non-limiting examples.
Example 1 Analysis of tissue distribution was performed by the Northern blotting technique using commercially available Blots of human RNA (Human Multiple Tissue Northern Blots I, II, and III; and Master Dot Blots; Clontech Laboratories, Inc., Palo Alto, CA). A probe was obtained by restriction digest of a human zalpha33 clone with EcoRI and PvuII, resulting in two cDNA fragments of 288 by and 326 bp. The reaction mixture was electrophoresed on a 2% agarose gel, and the corresponding bands were excised and purified using commercially available gel purification reagents and protocol (QIAEX~ II gel extraction kit; Qiagen, Valencia, CA). The purified DNA
was radioactively labeled with 32P using a commercially available kit (RediprimeTM II
2 o random-prime labeling system; Amersham Corp., Arlington Heights, IL). The probe was purified using a commmercially available size exclusion column (NucTrap~
column; Stratagene, La Jolla, CA). A commercially available hybridization solution (ExpressHybTM Hybridization Solution; Clontech Laboratories Inc., Palo Alto, CA) was used for hybridization and prehybridization. The final hybridization solution contained 2 5 8 ml ExpressHybTM solution, 80 ~.l sheared salmon sperm DNA ( 10 mg/ml;
obtained from 5 Prime-3 Prime, Boulder, CO) and 1.6 x 107 cpm labeled probe.
Hybridization took place overnight at 55°C. After hybridization the blots were washed in 2X SSC, 0.1 % SDS at room temperature; then in 2X SSC, 0.1 % SDS at 60°C
followed by a O.1X
SSC, 0.1 % SDS wash at 60°C. The blots were exposed to film (Biomax AR film, 3 o Eastman Kodak Co., Rochester NY) for three days and developed.
One major transcript size was observed on the Human Multiple Tissue blots at ~ 1.Okb in all tissues. The signal was strongest in heart, liver, pancreas, testis, ovary, and thyroid tissue, with other tissues showing moderately lower expression levels. Signals on the Master Dot blot were present in all tisues with slightly higher 3 5 signals in aorta, testis, pituitary gland, thyroid gland, mamary gland, thymus, lung, fetal thymus, and fetal lung. Expression was also seen in blots of THPl and U937 monocyte cell lines.

Example 2 An expression plasmid encoding full-length mouse zalpha33 was constructed using the expression vector pEZE2. The vector pEZE2 was derived from pDC312 by the addition of additional restriction enzyme recognition sites to the multiple cloning site. pDC312 and pEZE2 contain an EASE segment, as described in WO 97/25420, which can improve expression of recombinant proteins two- to eight-fold in mammalian cells (e.g., Chinese Hamster Ovary (CHO) cells). The pEZE2 expression unit contains the CMV enhancer/promoter, the adenovirus tripartite leader sequence, a multiple cloning site for insertion of the coding region for the recombinant protein, an encephalomyocarditis virus internal ribosome entry site, a coding segment for mouse dihydrofolate reductase, and the SV40 transcription terminator. In addition, pEZE2 contains an E. coli origin of replication and a bacterial beta-lactamase gene.
A DNA fragment encoding zalpha33 with a C-terminal Glu-Glu tag (zalpha33CEE) was generated by PCR using a commercially available kit (Advantage~
2 PCR Kit, Clontech, Palo Alto, CA). The fragment included 5' FseI and 3' AscI
sites for direct cloning into the expression vector. The 5' primer contained an FseI
site, a Kozak sequence, and the first 31 base pairs of the native leader sequence for zalpha33.
The 3' primer contained the last 18 base pairs of zalpha33, a Glu-Glu tag sequence, a stop codon, and an AscI site. The PCR mixture included lul of template (plasmid containing the full-length mouse zalpha33 sequence). The reaction was run at 94°C, 1 minute; then 25 cycles of 94°C, 30 seconds; 55°C, 30 seconds;
68°C, 1 minute; then a final extension at 72°C for 7 minutes. The PCR-generated fragment was purified using a commercially available kit (QiaquickTM PCR Purification Kit, Qiagen Inc., Valencia, 2 5 CA) and digested with restriction enzymes AscI and FseI (New England Biolabs, Beverly, MA) in a single, 100-~,1 reaction. Five micrograms of the expression vector pEZE2 were also digested with FseI and AscI in a single, 100-~1 reaction. The digested DNA was fractionated by agarose gel electrophoresis, and the DNA fragments were isolated and purified using a commercially available kit (QiaquickTM Gel Extraction 3 0 Kit, Qiagen Inc.).
Five microliters of the zalpha33CEE DNA fragment and 1 u1 of the pEZE2 vector fragment were ligated overnight at room temperature using T4 DNA
ligase (high concentration) and reaction buffer obtained from New England Biolabs, Beverly, MA. One microliter of the ligation mixture was added to 25 ~,1 of 3 5 electrocompetant E. coli strain DH l OB (Life Technologies) in a 0.2-cm cuvette. The mixture was electroporated (BioRad E. coli Pulser) at 2.3 kv. To the cuvette, 1 mL of LB broth was added, and 100 ~,1 of the mix was plated onto LB/Ampicillin agar plates.

The plates were incubated overnight at 37°C, and 16 isolated colonies were picked for DNA mini prep using a commercially available kit (obtained from Qiagen Inc.).
Individual clones were screened by PCR for the presence of zalpha33CEE DNA, using the above-mentioned primers. DNA sequencing was performed on clones #1-6, to verify the correct full-length sequence. One clone contained the correct expected sequence, from which DNA was prepared using a commercially available kit (Qiagen~
Plasmid Maxi Kit, Qiagen Inc.). The plasmid was designated pKF0248.
Example 3 Plasmid pKF0248 was prepared for transfection into Chinese hamster ovary (CHO) cells. To insure sterility a single ethanol precipitation step was performed by combining 200 pg of plasmid DNA with 20 p1 of lOmg/ml sheared salmon sperm carrier DNA (5' ~ 3' Inc. Boulder, CO), 22 ~,1 of 3M sodium acetate (pH 5.2), and 484 p,1 of 100% ethanol (Gold Shield Chemical Co., Hayward, CA) and incubating the mixture on ice for 25 minutes. After incubation, the mixture was spun at 14,000 RPM
in a microcentrifuge at 4°C, the supernatant was removed, and the pellet was washed twice with 0.5 ml 70% ethanol and allowed to :.~r dry until it appeared as an opaque white color.
Protein-free and serum-free suspension-adapted CHO DG44 cells 2 0 (Chasm et al., Som. Cell. Molec. Genet. 12:555-666, 1986) were taken from a frozen stock (passage 36) and preparerd for transfection by culturing in PFCHO media (JRH
Biosciences, Lenexa, KS), 4 mM L-Glutamine, (JRH Biosciences), and lx HT
Supplement (Life Technologies Lot # 1024782) at 37°C and 5% CO~ in shake flasks at 120 RPM on a rotating shaker platform. The cells were allowed to recover from the 2 5 process of thaw before being transfected at passage 38 with the plasmid zalpha 33 Mouse-CEE/pEZE2 (PKFO 248 linearized with Fsp) by High Copy Electroporation.
The CHO DG44 cells were transfected by electroporation. While the DNA pellet was drying, 10e6 total cells (16.5m1) were spun in a 25 ml conical centrifuge tube at 900 RPM for 5 minutes. The cells were resuspended into a total volume of 300 ~l in PFCHO media as above and placed in an electroporation cuvette with a 0.4 cm electrode gap (Gene Pulser~ Cuvette; Bio-Rad Laboratories, Inc., Hercules, CA). After approximately 50 minutes of drying time the plasmid DNA
was resuspended into 500 ~1 of PFCHO growth media and added to the cuvette so that the total volume did not exceed 800 microliters. The mixture was allowed to sit at room 3 5 temperature for 5 minutes to decrease bubble formation. The cuvette was placed in an electroporator (Gene Pulser~ II; Bio-Rad Laboratories, Inc.) set at 0.296 kV
and 0.950 HC (high capacitence) and electroporated immediately with an actual reading of 0.310 kV and a time constant of 15.7 milliseconds.
The cells were allowed to sit 5 minutes at room temperature to recover before placement in 20 ml total volume of PFCHO media in a tissue culture flask using a sterile glass 9-inch transfer pipet previously baked for 4 hours at 260°C. The flask was placed at 37°C and 5% COZ for 48 hours, at which time the cells were counted by hemocytometer utilizing trypan blue exclusion and put into PFCHO selection media without HT supplement and containing 200 mM methotrexate (Cal Biochem; San Diego, CA).
Upon recovery from the selection process conditioned media containing the secreted zalpha 33 protein is assayed by Western Blot. Neat 72-hour conditioned media is diluted 1:2 in reducing sample buffer (NuPAGETM 2X buffer; Novex, San Diego, CA) containing 100 mM DTT (ICN Biochemicals, Costa Mesa, CA), and 25-~1 samples are loaded into appropriate wells of a 1.0 mM x 12 well, 4-12% Bis-Tris Gel (NuPAGETM; Novex) and run at 150 volts for approximateley one hour. The gel is run with a protein standard (Glu-Glu tagged leptin produced in Pichia methanolica) is serially diluted ranging from 100 ng down to 6.25 ng total per lane and 10 p1 of commercially available molecular weight markers (SeeBlueTM; Novex). Proteins are transferred to a 0.2 pm nitrocellulose membrane (Novex) at 30 volts for approximately 2 0 one hour. The blot is blocked in 10% nonfat dry milk (Carnation) in Western A buffer (0.25% gelatin, SOmM Tris-HCl pH 7.4, 150 mM NaCI, SmM EDTA, 0.05%
octylphenylpolyethylene glycol (Igepal~-CA630)) either overnight at 4°C
or for one hour at room temperature on a rotating shaker platform. Thirty ml of mouse anti-Glu-Glu monoclonal antibody (BabCO Berkeley Antibody Company, Richmond, CA) diluted to 0.1 ~.g/ml in 2.5% nonfat dry milk in Western A is overlayed on the membrane, and the blot is incubated at room temperature on a rotating platform for one hour. The antibody solution is then discarded, and the membrane is rinsed three times (about 50 ml each time) with Western A. The blot is then incubated with 30 ml of goat anti-mouse IgG-HRP secondary antibody (1 mg/ml; Santa Cruz Biotechnology, Inc., 3 0 Santa Cruz, CA) at a 1:2000 dilution under the same conditions for one hour. The secondary antibody solution is discarded and the membrane rinsed as described above.
protein is detected by chemiluminescence using commercially available reagents (ECLTM Plus Western Blotting Detection System; Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and inctrumentation (Lumi-ImagerTM F1; Roche Molecular 3 5 Biochemicals, Indianapolis, IN).
Example 4 The protein coding region of mouse zalpha33 was amplified by PCR
using primers that added FseI and AscI restriction sites at the 5' and 3' termini respectively. PCR primers ZC23310 (SEQ ID N0:8) and ZC23311 (SEQ ID N0:9) were used with template plasmid containing the full-length mouse zalpha33 cDNA
in a 5 PCR reaction as follows: one cycle at 95°C for 5 minutes; followed by 15 cycles at 95°C for 0.5 min., 58°C for 0.5 min., and 72°C for 0.5 min.; followed by 72°C for 10 min.; followed by a 4°C soak. The PCR reaction product was loaded onto a 1.2% (low melt) agarose (SeaPlaque~ GTG; FMC Corp, Rockland, ME) gel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). The zalpha33 PCR product was excised from the gel.
10 The gel slice was melted at 65°C, and the DNA was extracted twice with an equal volume of Tris-buffered phenol and EtOH precipitated. The DNA was resuspended in 10 ~1 dH20, digested with FseI-AscI, phenol/chloroform extracted, EtOH
precipitated, and rehydrated in 20 p1 TE (Tris/EDTA pH 8). The 537-by zalpha33 fragment was then ligated into the FseI-AscI sites of a modified pAdTrack CMV (He et al., Proc.
15 Natl. Acad. Sci. USA 95:2509-2514, 1998). This construct also contained the green fluorescent protein (GFP) marker gene. The CMV promoter driving GFP expression was replaced with the SV40 promoter, and the SV40 polyadenylation signal was replaced with the human growth hormone polyadenylation signal. In addition, the native polylinker was replaced with FseI, EcoRV, and AscI sites. This modified form 2 0 of pAdTrack CMV was named pZyTrack. Ligation was performed using a DNA
ligation and screening kit (Fast-LinkT""; Epicentre Technologies, Madison, WI). Clones containing the zalpha33 cDNA were identified by standard miniprep procedures.
To linearize the plasmid, approximately 5 p,g of the pZyTrack zalpha33 plasmid was digested with PmeI. Approximately 1 pg of the linearized plasmid was cotransformed 25 with 200 ng of supercoiled pAdEasy (He et al., ibid.) into BJ5183 cells using a Bio-Rad Gene Pulser at 2.SkV, 200 ohms, and 25p.Fa. The entire co-transformation was plated on 4 LB plates containing 25 ~,g/ml kanamycin. The smallest colonies were picked and expanded in LB/kanamycin, and recombinant adenovirus DNA was identified by standard DNA miniprep procedures. Digestion of the recombinant adenovirus DNA
3 0 with FseI-AscI confirmed the presence of the zalpha33 sequence. The recombinant adenovirus miniprep DNA was transformed into E. coli strain DH10BTM competent cells (Life Technologies, Gaithersburg, MD), and DNA was prepared by anion exchange chromatography using a commercially available plasmid isolation kit (QIAGEN~ Plasmid Maxi Kit; Qiagen, Inc., Valencia, CA).
3 5 Approximately 5 ~.g of recombinant adenoviral DNA was digested with PacI enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 ~.1 containing 20-30U of PacI. The digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol. The DNA pellet was resuspended in 5 ~1 distilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc., Montreal, Canada), inoculated the day before and grown to 60-70% confluence, was transfected with the PacI-digested DNA. The PacI-digested DNA
was diluted up to a total volume of 50 p1 with sterile HBS (150 mM NaCI, 20 mM
HEPES). In a separate tube, 25 p,1 of lmg/ml N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts (DOTAP) (Boehringer Mannheim, Indianapolis, IN) was diluted to a total volume of 100 ~.1 with HBS. The DNA was added to the DOTAP, mixed gently by pipeting up and down, and left at room temperature for 15 minutes.
The media was removed from the 293A cells, and the cells were washed with 5 ml serum-free MEMalpha containing 1mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and 25 mM HEPES buffer (media components obtained from Life Technologies, Gaithersburg, MD). 5 ml of serum-free MEM was added to the 293A
cells and held at 37°C. The DNA/lipid mixture was added drop-wise to the T25 flask of 293A cells, mixed gently, and incubated at 37°C for 4 hours. After 4 hours the media containing the DNA/lipid mixture was aspirated off and replaced with 5 ml complete MEM containing 5% fetal bovine serum. The transfected cells were monitored for GFP expression and formation of foci (viral plaques).
Seven days after transfection of 293A cells with the recombinant 2 0 adenoviral DNA, the cells expressed GFP and started to form foci. The crude viral lysate was collected with a cell scraper and transferred to a 50-ml conical tube. To release most of the virus particles from the cells, three freeze/thaw cycles were done in a dry ice/ethanol bath and a 37° waterbath.
The crude lysate was amplified (primary (1°) amplification) to obtain a 2 5 working "stock" of zalpha33 recombinant adenovirus (rAdV) lysate. Ten 10-cm plates of nearly confluent (80-90%) 293A cells were set up 20 hours in advance. 200 ml of crude rAdV lysate was added to each 10-cm plate, and the plates were monitored for 48 to 72 hours for CPE (cytopathic effect) under the white light microscope and expression of GFP under the fluorescent microscope. When all of the cells showed CPE, the 1 °
3 0 stock lysate was collected, and freeze/thaw cycles were performed as described above.
For secondary (2°) amplification, twenty 15-cm tissue culture dishes of 293A cells were prepared so that the cells were 80-90% confluent. All but 20 ml of 5%
MEM media was removed, and each dish was inoculated with 300-500 ml 1 °
amplified rAdv lysate. After 48 hours the cells were lysed from virus production. The lysate was 3 5 collected into 250-ml polypropylene centrifuge bottles.
To purify the rAdV, NP-40 detergent was added to a final concentration of 0.5% to the bottles of crude lysate in order to lyse all cells. Bottles were placed on a rotating platform for 10 minutes, agitating as fast as possible without the bottles falling over. The debris was pelleted by centrifugation at 20,000 X G for 15 minutes.
The supernatants were transferred to 250-ml polycarbonate centrifuge bottles, and 0.5 volume of 20% PEG-8000/2.5 M NaCI solution was added. The bottles were shaken overnight on ice. The bottles were centrifuged at 20,000 X G for 15 minutes, and supernatants were discarded into a bleach solution. Using a sterile cell scraper, the precipitate from 2 bottles was resuspended in 2.5 ml PBS. The virus solution was placed in 2-ml microcentrifuge tubes and centrifuged at 14,000 X G for 10 minutes to remove any additional cell debris. The supernatant from the 2-ml microcentrifuge tubes was transferred to a 15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/ml with CsCI. The volume of the virus solution was estimated, and 0.55 g/ml of CsCI
was added. The CsCI was dissolved, and 1 ml of this solution weighed 1.34 g.
The solution was transferred to polycarbonate thick-walled centrifuge tubes (3.2 ml) (Beckman #362305) and spun at 348,000 X G for 3-4 hours at 25°C in a Beckman Optima TLX microultracentrifuge with a TLA-100.4 rotor. The virus formed a white band. Using wide-bore pipette tips, the virus band was collected.
The virus preparation was desalted by gel filtration using commercially available columns and cross-liked dextran media (PD-10 column prepacked with Sephadex~ G-25M; Pharmacia, Piscataway, NJ). The column was equilibrated with 2 0 ml of PBS. The virus was loaded and allowed to run into the column. Five ml of PBS
was added to the column, and fractions of 8-10 drops were collected. The optical densities of 1:50 dilutions of each fraction were determined at 260 nm on a spectrophotometer. A clear absorbance peak was present between fractions 7-12.
These fractions were pooled, and the optical density (OD) of a 1:25 dilution was 2 5 determined. Virus concentration was calculated by the formula: (OD at 260nm)(25)(1.1 x 10'2) = virions/ml. The OD of a 1:25 dilution of the zalpha33 rAdV
was 0.059, giving a virus concentration of 2.8 X 10'2 virions/ml.
To store the virus, glycerol was added to the purified virus to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80°C.
3 0 A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Canada) was followed to measure recombinant virus infectivity. Briefly, two 96-well tissue culture plates were seeded with 1 X 104 293A cells per well in MEM
containing 2% fetal bovine serum for each recombinant virus to be assayed. After 24 hours, 10-fold dilutions of each virus from 1 X 10-2 to 1 X 10-~4 were made in MEM
containing 3 5 2% fetal bovine serum. 100 ~.1 of each dilution was placed in each of 20 wells. After 5 days at 37°C, wells were read either positive or negative for CPE and a value for plaque forming units/ml (PFU) was calculated.

TCIDS° formulation used was as per Quantum Biotechnologies, Inc., above. The titer (T) was determined from a plate where virus used was diluted from 10-2 to 10-~4, and read 5 days after the infection. At each dilution a ratio (R) of positive wells for CPE per the total number of wells was determined.
To calculate titer of the undiluted virus sample: the factor, "F" = 1+d(S-0.5); where "S" is the sum of the ratios (R); and "d" is LoglO of the dilution series, for example, "d" is equal to 1 for a ten-fold dilution series. The titer of the undiluted sample is T = 10«+~ = TCIDS°/ml. To convert TCIDS°/ml to pfu/ml, 0.7 is subtracted from the exponent in the calculation for titer (T).
The zalpha33 adenovirus had a titer of 3.2 X 10'° pfu/ml.
Example 5 Approximately 10 ~g pZytrack vector containing the sequence-confirmed mouse zalpha33 coding region was digested with FseI and AscI. The vector was then ethanol precipitated, and the pellet was resuspended in Tris-EDTA
buffer.
The released 537-by zalpha33 fragment was isolated by running the digested vector on a 1.2% agarose gel (SeaPlaque° GTG: FMC Corp., Rockland, ME) and excising the fragment. DNA was purified using a commercially available kit (QiaQuickTM Gel Extraction Kit, Qiagen Inc.).
The mouse zalpha33 fragment was then ligated into the vector pTGl2-8, which was previously digested with FseI and AscI. The pTG 12-8 plasmid, designed for expression of a gene of interest in transgenic mice, contains an expression cassette flanked by 10 kb of MT-1 (mouse metallothionein gene) 5' DNA and 7 kb of MT-1 3' DNA. The expression cassette comprises the MT-1 promoter, the rat insulin II
intron, a 2 5 polylinker for the insertion of the desired clone, and the human growth hormone poly A
sequence.
About one microliter of the ligation reaction mixture was electroporated into E. coli host cells (Electromax DH10BTM cells; Life Technologies) according to the supplier's directions, plated onto LB plates containing 100 ~g/ml ampicillin, and 3 o incubated overnight at 37°C. Colonies were picked and grown in LB
media containing 100 ~g/ml ampicillin. Miniprep DNA was prepared from the picked clones and screened for the mouse zalpha33 insert by restriction digestion with EcoRI and agarose gel electrophoresis. Maxipreps of the correct pTGl2-8 murine zalpha33 construct were performed.
3 5 A SaII fragment containing 5' and 3' flanking sequences, the MT
promoter, the rat insulin II intron, murine zalpha33 cDNA and the human growth hormone poly A sequence was prepared and used for microinjection into fertilized days at 37°C, wells were read either p mouse oocytes. Two transgenic mice with high zalpha33 expression levels had low white blood cell counts.
Example 6 Four confluent T-162 flasks of HaCaT human keratinocyte cells (Boukamp et al., J. Cell. Biol. 106:761-771, 1988) were trypsinized. Each pellet was resuspended in 4 ml DMEM + 5% FBS. The HaCat cells were transduced by adding purified mouse zalpha33 recombinant adenovirus (AdZy/zalpha33) or parental adenovirus at a MOI of 500 particles per cell and shaking slowly at 37°C for 1 hour.
to The cells were then transfered to two T-162 flasks each containing 30 ml growth media and incubated 48 hours (until confluent). The cells were rinsed twice with 1X
PBS and re-fed with 30 ml per flask serum-free/phenol-red-free DMEM containing L-glutamine, sodium pyruvate and HEPES buffer. The cells were incubated and harvested three times at 72-hour intervals. The collected conditioned media were centrifuged to remove cellular debris and concentrated to lOX using Millipore 80 ml, SK cut-off centrifugal filter devices. The concentrates were frozen in 1-ml aliquots at -80°C.
Example 7 Mouse zalpha33 was assayed in an aortic ring outgrowth assay (Nicosia 2 o and Ottinetti, Laboratory Investigation 63( 1 ):115, 1990; Villaschi and Nicosia, Am. J.
Pathology 143(1):181-190, 1993). Thoracic aortas were isolated from 1-2 month old SD female rats and transferred to petri dishes containing HANK's buffered salt solution. The aortas were flushed with additional HANK's buffered salt solution to remove blood, and adventitial tissue surrounding the aortas was carefully removed.
2 5 Cleaned aortas were transferred to petri dishes containing serum-free basal medium (EBM; Clonetics, San Diego, CA). Aortic rings were obtained by slicing approximately 1-mm sections using a scalpel blade. The ends of the aortas used to hold the aorta in place were not used. The rings were rinsed in fresh EBM basal media and placed individually in wells of a 24-well plate coated with basement membrane matrix 3 0 (Matrigel~; Becton Dickinson, Franklin Lakes, NJ). The rings were overlayed with an additional 50 ~ul of Matrigel and placed at 37°C for 30 minutes to allow the matrix to gel. Test samples were diluted in EBM basal serum-free media supplemented with units/ml penicillin, 100 ~g/ml streptomycin and HEPES buffer and added at 1 ml/well.
Background control was EBM basal serum-free media alone. Basic FGF (R&D
3 5 Systems, Minneapolis, MN) at 20 ng/ml was used as a positive control.
AdZy/zalpha33 adenovirus was added to wells, assuming a cell count of 500,000 cells and a multiplicity of infection of 5000 particles/cell. A null pZyTrack adenovirus (zPar) was used as a control. lOX conditioned media generated in HaCat keratinocyte cells by a 4 day tranduction with the AdZy/zalpha33 at an MOI of 500 particles per cell was also used in this assay. A control conditioned media generated using the zPar rAdenovirus to transduce HaCat cells was used as the matching negative control. Samples were 5 added in a minimum of quadruplets. Rings were incubated for 5-7 days at 37°C and analyzed for growth. Aortic outgrowth was scored by multiple, blinded observers using 0 as no growth and 4 as maximum growth. Zalpha33/HaCat conditioned media generated by transduction with the AdZy/zalpha33 produced a significant increase in outgrowth as compared to controls, and was comparable to other potent growth factors 10 (e.g., bFGF).
Example 8 Various human tissues, including heart, pancreas, testis, liver, ovary and appendix, were isolated and screened for zalpha33 expression by in situ hybridization.
15 The tissues were fixed overnight in 10°1o neutral phosphate-buffered formalin (Surgipath, Richmond, IL), and embedded in paraffin (Oxford Scientific, St.
Louis, MO) using standard techniques. Tissues were sectioned at 4 to 8 microns.
Tissues were prepared using a standard protocol ("Development of non-isotopic in situ hybridization" at http://dir.niehs.nih.gov/dirlep/ish.html). Briefly, tissue sections were 2 0 deparaffinized with a commercially available histological clearing agent HistoClearTM;
National Diagnostics, Atlanta, GA) and then dehydrated with ethanol. Next they were digested with 50 ~g/ml Proteinase K (Boehringer Diagnostics, Indianapolis, IN) at 37°C
for 2 to 5 minutes. This step was followed by acetylation and re-hydration of the tissues.
2 5 An antisense probe was generated from the zalpha33 sequence using Sp6 RNA polymerase. The probe was labeled with digoxigenin (Boehringer) using a commercially available in vitro transcription system (Promega, Madison, WI) as directed by the manufacturer.
In situ hybridization was performed with the digoxigenin-labeled 3 0 zalpha33 probe. The probe was added to the slides at a concentration of 1 to 5 pmol/ml for 12 to 16 hours at 57.5°C. Slides were subsequently washed in 2 X
SSC and 0.1 X
SSC at 55°C. The signals were amplified using a tyramide signal amplification kit (TSATM Signal Amplification; NEN Life Science Products, Inc., Boston, MA) (see, US
Patents Nos. 5,731,158; 5,583,001; and 5,196,306) and visualized with a commercially 3 5 available substrate kit (Vector~ Red; Vector Laboratories, Burlingame, CA) as directed by the manufacturer. The slides were then counter-stained with hematoxylin (Vector Laboratories).

Signals were seen in the heart, pancreas, testis, liver, and appendix. The positive-staining cells appeared to be endothelial cells of vessels, immune cells, acinar cells of pancreas, and spermatocytes of testis.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE LISTING
<110> ZymoGenetics, Inc.
<120> HELICAL CYTOKINE ZALPHA33 <130> 99-38 <150> 09/333,076 <151> 1999-06-14 <160> 10 <170> FastSEQ for Windows Version 3.0 <210>1 <211>954 <212>DNA

<213>Homo Sapiens <220>
<221> CDS
<222> (136)...(672) <400> 1 gccgctgttt tgaaatcggg ccgcgggggg tctctcaagc tggttccaac gctgaggccc 60 cacagcctcc caattccggg cagacccctg acacctgctg tctggcccct tccggcctga 120 agctgcagcc gcgcc atg tcc acc cct ccg ttg gcc gcg tcg ggg atg gcg 171 Met Ser Thr Pro Pro Leu Ala Ala Ser Gly Met Ala ccc ggg ccc ttc gcc ggg ccc cag get cag cag gcc gcc cgg gaa gtc 219 Pro Gly Pro Phe Ala Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu Val aac acg gcg tcg ctg tgc cgc atc ggg cag gag aca gtg cag gac atc 267 Asn Thr Ala Ser Leu Cys Arg Ile Gly Gln Glu Thr Val Gln Asp Ile gtg tac cgc acc atg gag atc ttc cag ctc ctg agg aac atg cag ctg 315 Val Tyr Arg Thr Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln Leu cca aat ggt gtc act tac cac act gga aca tat caa gac cgg tta aca 363 Pro Asn Gly Val Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr aag cta cag gat aat ctt cgc caa ctt tca gtt ctc ttc agg aag ctg 411 Lys Leu Gln Asp Asn Leu Arg Gln Leu Ser Val Leu Phe Arg Lys Leu aga ttg gta tat gac aaa tgc aat gaa aac tgt ggt ggg atg gat ccc 459 Arg Leu Val Tyr Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro att cca gtc gag caa ctt att cca tat gtg gaa gaa gat ggc tca aag 507 Ile Pro Val Glu Gln Leu Ile Pro Tyr Val Glu Glu Asp Gly Ser Lys aat gat gat cgg get ggc cca cct cgt ttt get agt gaa gag agg cga 555 Asn Asp Asp Arg Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg gaa att get gaa gta aat aaa aaa ctc aaa cag aag aat caa cag ctg 603 Glu Ile Ala Glu Val Asn Lys Lys Leu Lys Gln Lys Asn Gln Gln Leu aaa caa att atg gat caa tta cga aat ctc atc tgg gat ata aat gcc 651 Lys Gln Ile Met Asp Gln Leu Arg Asn Leu Ile Trp Asp Ile Asn Ala atg ttg gca atg agg aac taa gctgatattt aaatttcctg ctttacacat 702 Met Leu Ala Met Arg Asn gttataccattgttttttccctcaagtattttttccctgtgaagaagattatttatctgc762 ttttattttagtcactaaaactaaagtttttatttttacattgtgatttttacattaaaa822 tattaacttttttaatgctattttatgaaagattattgtaataaactttgatggggtttg882 tattttggttaatcttcatgaattgaataattgtttttttaaagcaaaataaagtttttt942 aaataaatgtto 954 <210> 2 <211> 178 <212> PRT
<213> Homo sapiens <400> 2 Met Ser Thr Pro Pro Leu Ala Ala Ser Gly Met Ala Pro Gly Pro Phe Ala Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu Val Asn Thr Ala Ser Leu Cys Arg Ile Gly Gln Glu Thr Val Gln Asp Ile Val Tyr Arg Thr Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln Leu Pro Asn Gly Val Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr Lys Leu Gln Asp Asn Leu Arg Gln Leu Ser Val Leu Phe Arg Lys Leu Arg Leu Val Tyr Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro Ile Pro Ual Glu Gln Leu Ile Pro Tyr Val Glu Glu Asp Gly Ser Lys Asn Asp Asp Arg Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg Glu Ile Ala Glu Val Asn Lys Lys Leu Lys Gln Lys Asn Gln Gln Leu Lys Gln Ile Met Asp Gln Leu Arg Asn Leu Ile Trp Asp Ile Asn Ala Met Leu Ala Met Arg Asn <210>3 <211>950 <212>DNA

<213>Mus musculus <220>
<221> CDS
<222> (120)...(656) <400> 3 cactgttggc ctactggagt cttccggtac agcgcttgca tcgcggcggg cggaagtggc 60 gccgcttttt tgaaatcggc cgagtgggct cgcgccggac ccgagccgcc gggggtgcc 119 atg tcc acc cct ccg ctg gcg ccc acg ggc atg gcg tcc ggg ccc ttc 167 Met Ser Thr Pro Pro Leu Ala Pro Thr Gly Met Ala Ser Gly Pro Phe ggc ggc ccg cag get cag cag gcc gcg cgc gag gtc aac acg gcc acg 215 Gly Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu Val Asn Thr Ala Thr ctg tgc cgc atc ggg cag gag acc gtg cag gac atc gtg tac cgc acc 263 Leu Cys Arg Ile Gly Gln Glu Thr Val Gln Asp Ile Val Tyr Arg Thr atg gag atc ttc cag ctg ctc agg aac atg cag ctg cca aat ggt gtc 311 Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln Leu Pro Asn Gly Val act tac cat act gga act tac caa gac cgg cta aca aag ctg cag gac 359 Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr Lys Leu Gln Asp cac ctt cgg caa ctt tct att ctc ttc agg aag ctg cga ctg gtc tat 407 His Leu Arg Gln Leu Ser Ile Leu Phe Arg Lys Leu Arg Leu Val Tyr gac aaa tgt aat gag aac tgt ggt ggg atg gac ccc att cct gtt gag 455 Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro Ile Pro Ual Glu caa ctg att cca tat gtg gat gaa gat ggc tca aag aac gac gac cgg 503 Gln Leu Ile Pro Tyr Val Asp Glu Asp Gly Ser Lys Asn Asp Asp Arg get ggt cca cct cgt ttt get agc gaa gag aga cga gaa att gta gaa 551 Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg Glu Ile Ual Glu gta aat aag aaa ctc aaa cag aag aat caa cag ctg aag cag att atg 599 Val Asn Lys Lys Leu Lys Gln Lys Asn Gln Gln Leu Lys Gln Ile Met gat caa tta cgg aat ctc atc tgg gac ata aat gcc atg ctg gca atg 647 Asp Gln Leu Arg Asn Leu Ile Trp Asp Ile Asn Ala Met Leu Ala Met agg aac taa agcgatattt aaatctcctg ctctactcat gtgatgaagt 696 Arg Asn tgggtttttcccccctcttgagtattcctccctttgaaaaacgttattta tgtctttatt756 ttaacagctagcactaaagtttctgttttcactttaaagtatttactagc ttttttttta816 atactgtgggttttatgaaagattattgtaatacctttgatagggtataa attttggtta876 atcttcagaaattgaataaattaaaaaatacaaataaaaatttaaaaaaa aaaaaaaaaa936 aaaggccacatgtg 950 <210>4 <211>178 <212>PRT

<213>Mus musculus <400> 4 Met Ser Thr Pro Pro Leu Ala Pro Thr Gly Met Ala Ser Gly Pro Phe Gly Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu Val Asn Thr Ala Thr Leu Cys Arg Ile Gly Gln Glu Thr Val Gln Asp Ile Val Tyr Arg Thr Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln Leu Pro Asn Gly Val Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr Lys Leu Gln Asp His Leu Arg Gln Leu Ser Ile Leu Phe Arg Lys Leu Arg Leu Ual Tyr Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro Ile Pro Val Glu Gln Leu Ile Pro Tyr Val Asp Glu Asp Gly Ser Lys Asn Asp Asp Arg Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg Glu Ile Val Glu Val Asn Lys Lys Leu Lys Gln Lys Asn Gln Gln Leu Lys Gln Ile Met Asp Gln Leu Arg Asn Leu Ile Trp Asp Ile Asn Ala Met Leu Ala Met Arg Asn <210> 5 <211> 178 <212> PRT
<213> Artificial Sequence <220>
<223> variant polypeptides <221> VARIANT
<222> (41)...(45) <223> Xaa is Leu, Ile. Val, Met, Phe, Trp, Gly, or Ala <221> VARIANT
<222> (48)...(48) <223> Xaa is Thr. Leu, Ile. Val, Met, Phe, Trp, Gly, or A1 a <221> VARIANT
<222> (51)...(78) <223> Xaa is Leu, Ile, Val, Met, Phe, Trp. Gly, or Ala <221> VARIANT
<222> (81)...(81) <223> Xaa is Asn, Leu. Ile, Ual, Met. Phe, Trp, Gly, Ala, or His <221> VARIANT
<222> (82)...(118) <223> Xaa is Leu. Ile, Val, Met, Phe, Trp, Gly, or Ala <221> VARIANT
<222> (121)...(121) <223> Xaa is Asp, Leu, Ile, Val, Met, Phe, Trp, Gly, or Ala <221> VARIANT
<222> (122)...(122) <223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, or Ala <221> VARIANT
<222> (125)...(125) <223> Xaa is Asn, Leu, Ile, Val, Met, Phe, Trp. Gly, or Ala WO 00/77209 PCT/~JS00/16441 <221> VARIANT
<222> (149)...(149) <223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, or Ala <221> VARIANT
<222> (152)...(152) <223> Xaa is Lys, Leu, Ile, Val, Met, Phe, Trp, Gly, or Ala <221> VARIANT
<222> (153)...(153) <223> Xaa is Asn, Leu, Ile, Val, Met, Phe, Trp, Gly, or Ala <221> VARIANT
<222> (156)...(163) <223> Xaa is Leu, Ile, Ual, Met: Phe, Trp, Gly, or Ala <400> 5 Met Ser Thr Pro Pro Leu Ala Ala Ser Gly Met Ala Pro Gly Pro Phe Ala Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu Val Asn Thr Ala Ser Leu Cys Arg Ile Gly Gln Glu Thr Xaa Gln Asp Xaa Xaa Tyr Arg Xaa Met Glu Xaa Xaa Gln Leu Xaa Arg Asn Met Gln Leu Pro Asn Gly Val Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr Lys Xaa Gln Asp Xaa Xaa Arg Gln Xaa Ser Ual Xaa Xaa Arg Lys Xaa Arg Leu Val Tyr Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro Ile Pro Xaa Glu Gln Xaa Xaa Pro Tyr Xaa Glu Glu Xaa Xaa Ser Lys Xaa Asp Asp Arg Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg Glu Ile Ala Glu Val Asn Lys Lys Xaa Lys Gln Xaa Xaa Gln Gln Xaa Lys Gln Xaa Xaa Asp Gln Xaa Arg Asn Leu Ile Trp Asp Ile Asn Ala Met Leu Ala Met Arg Asn <210> 6 <211> 534 <212> DNA
<213> Artificial Sequence <220>
<223> degenerate sequence <221> misc_feature <222> (1). .(534) <223> n = A,T,C or G
<400> 6 atgwsnacnccnccnytngcngcnwsnggnatggcnccnggnccnttygcnggnccncar 60 gcncarcargcngcnmgngargtnaayacngcnwsnytntgymgnathggncargaracn 120 gtncargayathgtntaymgnacnatggarathttycarytnytnmgnaayatgcarytn 180 ccnaayggngtnacntaycayacnggnacntaycargaymgnytnacnaarytncargay 240 aayytnmgncarytnwsngtnytnttymgnaarytnmgnytngtntaygayaartgyaay 300 garaaytgyggnggnatggayccnathccngtngarcarytnathccntaygtngargar 360 gayggnwsnaaraaygaygaymgngcnggnccnccnmgnttygcnwsngargarmgnmgn 420 garathgcngargtnaayaaraarytnaarcaraaraaycarcarytnaarcarathatg 480 gaycarytnmgnaayytnathtgggayathaaygcnatgytngcnatgmgnaay 534 <210> 7 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> peptide tag <400> 7 Glu Tyr Met Pro Met Glu <210> 8 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC23310 <400> 8 cacacaggcc ggccaccatg tccacccctc cgctggcgc 39 <210> 9 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC23311 <400> 9 cacacaggcg cgcctttagt tcctcattgc cagcatggc 39 <210>10 <211>20598 <212>DNA

<213>Homo Sapiens <400>

ctcaacaatcattcaataagtatttattgatacttgtgccaatcactaggtgttgagaat 60 aagtgaataaaatagtctgttttcatggagttggcagtgtgtgtagatagggtgagataa 120 agtctgaaagtaaacaaactgaaagctatataaaatatcagacagtaggccgggcgcggt 180 ggcttatgcctgtaatcccagcactttgggaggccgaggcgggcggatcacgaggtcagg 240 agatcgagaccatcccggctaacacggtgaaaccccgtctctactaaaaatacaaaaaat 300 ttgtcgggcgtggtggcgggcgcctgtagtcccagctactcgggaggccgaggctggaga 360 atggtgtgaacccgggaggcggagcttgcagtgagccgaggtcgcgccactgcactccag 420 cctgggtgacagagcgagactccatctcaaaaagaaaaagaaaaaaaaatcagacactaa 480 taagagccagagtcagaaagaagtgtgtgtgttgatcattttttttgcttacagatactg 540 ggaaaactcaaagaataaaacatctgagcagggaccaagagcaatcggggagtccgtatc 600 atgtcacagagaagcgtacaagcttaggaatcttgttttctgaattcgaatcgcaggttg 660 ccagttacacctgtatgcgactcaacaagtactttaacctgtctcttagctgttatacta 720 caaacctcaggcttgttaagagaaaatgctccgcaaggaactaaacacagaaggttctaa 780 cgctgagccaagactgggaaacgaactctgggaactcaccccaggctccccaagaacatc 840 gcccctctggctggagcgcaattggtgattggctacttaacccgtccgtcctttcccgcc 900 caggggtccaatccaatccagcccggctccgctcggagacagttcgccgagtgggcggtg 960 tctatgacgttttctgacgtgttacgtcacagtgggcggaagtcgcggccgctgttttga 1020 aatcgggccgcggggggtctctcaagctggttccaacgctgaggccccacagcctcccaa 1080 ttccgggcagacccctgacacctgctgtctggccccttccggcctgaagctgcagccgcg 1140 ccatgtccacccctccgttggccgcgtcggggatggcgcccgggcccttcgccgggcccc 1200 aggctcagcaggccgcccgggaagtcaacacggcgtcgctgtgccgcatcgggcaggaga 1260 cagtgcaggacatcgtgtaccgcaccatggagatcttccagctcctgaggaacatgcagg 1320 WO 00/77209 PCT/~JS00/16441 taggaaggcgggcgcgcgaggccagggggatgcagctgggagggaaagggcctctggttt1380 cctctttacgtggggcccgtggatgtcatcggggctgccctgggcaaaccttaggtgtag1440 ggtctcccccattcacactcaagagtgtccccaaaagtcggatttgtagaggtgatgttt1500 tccagaccagatttctgcagaattgaagccaattttttttagattcaatcgagctcttta1560 gttactctgtaaactcttgcacctttcaaggcagagatgggtcgcactccacttccctct1620 ttaggttgctttaaatgttttttccagcatttcttctggggaggtccaaggagttgaaga1680 atttacattttttttaaaaaaacagtcctcctttttttggcatagttaatatacagtaaa1740 ctacactcacctaaagttttgatatatatgtgtactgtagtctgtatacttgtgaaatta1800 tcaccacaatcaaaatagtgtacacatcatcccagaaagtttcttcatacccctctgaca1860 tctttgcttcccaccttccatcgctaagcaaccattgatctgcgttctgtaatcataaat1920 taatttgcatttgctagggttttatataaacggagtctacagtatgtgggcttttttttt1980 tgtctgagatccttcactaagaatgcatcaataatttcctttttattgctgagtaatgtt2040 ccattgtatccatgtatcaccatttgcctaccttttcttctgttgatggacatttgggtt2100 gtttccactttttggctgttgtaaataatgctgctatgaacttttgctgacaaatttttg2160 tgtgaacgtatgtgtcatttctcttggacagattcaggagtggaattgctggatcatgca2220 gtacatgtctgtttaatactttaagaaacttttccaaagtggcctgagccattttacgtc2280 ccgattattgctagtgggaatgtaaacattgctgtttgagggttctagtttctcctcatc2340 ctcatcaacacctgttatgtcttgatgctttccaaatacttctcccagtctatcctgcct2400 ttttattctcttaatactgtcttttgaagaacagacattttaaattttgatggaatccag2460 cttactgatttgttcttttatagattttggttttgctgtcatacctaagaaatctttgcc2520 taacccaaggtgacaaagggtttctgttttcttctagaagttttgtactttcaagcttta2580 tatttaggtctacaacctatatttagttttgtgagtttttaaatatgtatacatagtggc2640 agatacggatccaagttcattgttttgcatttggatatccagttgttccagcaccatttt2700 gctgacccatttttaatgcttgcttatattacccactacatgtagtagatattatttttg2760 ttttacagatgaagattctgtacttcagagagataaagtgacttgcccaaacctagtcag2820 tggtagggtaaatttgaaaccaagttttcaaaaatcttacagctcttatttctgtgggtg2880 tccaaaaaagtgttttattgctttttttttttttaactggtcaagactttttttttcgtc2940 ctgattataagagtaattcagtttcgtggaaatttttaagaccatgtgaaatatagtaca3000 aaggagatccaggagaaattatgacattctttggcaataatttaaaaaaatgaatagatt3060 catatctagaatgaataaagtacggattaaataatgtctcagaatttagcatactttaaa3120 aaaaattttgtcctgttatatgcaaagcaccgaataatacctaacacttacgaagtactt3180 tccttggtcaggtaccctgttataggagcagttttaaggagtatttttatccctatttta3240 cagtaaaggaaactgaggcttaatgagagaaaaaataaattacccaacatcacacacgac3300 taatagctcttcctactaacctctttttaatactaacttgctgtcaatgtacattattaa3360 aagatataatacaggttcatgttctacgcagaactaaactttttcaaaaaaagtatttta3420 tcaataactagattacatgtggacctgcattcaaagccaacttttttttttttaagtttt3480 ttttttttgtctgtaagtttattcaatgcaaaataatcctctccaattttactgaggtgg3540 ctgaccatgtccacgaccaaatccgcctctaaactggaattcggttgttgacccagcccc3600 agtctcggctttcttgtcggcaccagggggcacagcactccgtctgtaggtatctctgtc3660 ggcttcccctcttgtgagtcttgcaggtcgctcaccctccagacctttaggctgaggcct3720 gccagtctctggacggctgcggcgtagggtggcaggaacaatctccgggggcagatgaag3780 gtaatcacagagatactggataccctcattggtaaggtaccagtagaaatgtctccaggc3840 aaactgttccttcacgtagcctcaggacttgagaaactgcatggccttcatgacatgaag3900 gttggacacattcttgtctgccagctccgggtgcttaggcatgtggaatcctccttggcc3960 accatgattccctccttaaaaaggagttcataggctaggcgcggtggctcacgcctgtaa4020 tcccagcactttgagaggccgaggtgggcggatcacgaggtcaggaattcgagaccagcc4080 tagccaacatggtgaaaccccatctctactaaaaatacaaaaattagccgggcgtggtgg4140 cgggcgcctgtaatcccagctactcgggaggctggggcaggagaatcgcttgaacccggg4200 aggcggaggtggcagtgagccgagagatcgcgccactgcactccaggctgggcgacagag4260 cgagactctgtctcaagaaaaaggagttcataaatggcaatacgtttcttcttaagcttc4320 aacatttcggccactgtaggtctgggacccaaagccagcttttgagggactatgataatt4380 atatgtgtgcttttcattttgtgaaatctgtgatcactcatccaggacctttggggccta4440 tcataattggctgaaaagataacaatgggctccttcatttccaaaaaaactagaagaggt4500 gatagaaataggatattcttaactatatataacctagtgatttgttaatacatttttagt4560 attcacattttaaagtaatcgtctgtattattgatctacgagagtaaagtgctgtgaata4620 caaactgctaagaatttttctaagcatttgttttaaagaagtggttcctggtatacacta4680 gatgtccgttagccacttgagagtctgatgaaaactgtgagctctcttctgagagaaata4740 cacatacattttgaaaatttcaggggttaaaggaagatttaggatactccccaagtatcc4800 ttaaatctcagattaagagtccttgtggaagataccattgcttttgttctgtttattatt4860 ctttgaatctttaatttttaaaatacatagtacattcagaatttaggaataaaaaaggac4920 agtgcagtgaacattttccttcccattcctatctcttatactatgtacctaactcctctc4980 cacaggcagtgattgtgactgattcttactatttttgtgtatctttccagatattctctg5040 catattaagcaaatgtttatgtatattgtttcattttcgccttttttgcacagataatag5100 tatattatacacaaccattttgtaccttgcttttttcagttgatatattttggaatttat5160 tcatacccatacagaaaatactcccttatcctttttatagtgtatagtttacactgcatg5220 gaaatactgtaatttattaaaccagttctctagtgagggcatttggctgtttttcatctt5280 tccctgttaggctgcaccaaataacctcctacacaaatttgcacgtatacctgtcggata5340 cgtttttagaaatagctatgtgcttttgtaattagatacattttgatgaattgtcctcca5400 cagagattgaactaatttatgctacggcctaagaccagcacagtgagttatagctttttt5460 ggatctttgatcacttgataggtgaaaattctactttacatttctcctagtttgactgag5520 tttgaacatctcttaaaatattttaattatttatatttttttcctgctaaatgcttgttc5580 acttattattcacccatttgtctgtctttttttttttttcatcttttttacctaccttaa5640 ttgtaggatttagctatttacatactagagaaacctgctctgtgtgatacaacttaaagt5700 tatttttttccctgtcttttaccttttggctctgctaatggtagttttgccatgcatttt5760 ttttaagtagttgaatgtattagtcttttaggacttctaggccttatgcctagttagaaa5820 ggtcttccccactcaaattaggaagaattctataatttttaaaatttcagcctttacatt5880 taaaactgtgatccttatggaatttatcctataaaatgtgaggtctgggcctagccttaa5940 ctttttcaaaaggttcactgaataattcacttgcttcttccagtgatttcaggtagcttt6000 tatataagtgaatttgtttctggatgttctattctattccacgtagctgcctatctttta6060 tgtattaatgcaaaacattcttttaatttgtcaggatctaaatgtgttacacttattttt6120 cagaatttccctggctattcttgcttatttttccacatgtattttagaatcagcttattc6180 taagaaaaagcctgttagcatttttattaggtccgttcggcctagaaagacttgacatca6240 ttatgatgttgactcttactaaccaagaacatgcagtattctttttctgttttcctataa6300 ggtatcttaagttttcatttttatcttgagattttaaaagtttttttctaggtattttat6360 cttttctgttgccattgtaaatggggtcttcttttcccatgtttttgtttgtgttataga6420 tgaaaacagagctattgctttctctttggacctacttccttattgaattcttttattttt6480 agtagcactttatcatattatttgtaaatagattatgatgctttctttttaatgtttcaa 6540 catttttttctctttcttctctaattgcattggctagtgcttccagaacagtgttcactt 6600 acagaattagggtcttagtagacatccttttctgtttctgatcttagttggaatgcttct 6660 aatgattccacattaatcatagtgctgactctttaactgaaataaatgtatgttgtcaca 6720 tattgaggattttttattgagttttatattgattttttaaatgggtgttgaatttagttg 6780 aatgccttcttggtgtctatggatacgatcatgtatttatgggttttcatccatactcat 6840 gagtgagatggttattgatgagcttttcagctgaacagctacaccgtggtatataccaag 6900 ggatcttaggcatttttaccgtgtatactctgtactatggaaaccttgtctctctgatgt 6960 ttactgctctatcctagcttctagaacagtgctgacacatgaaacgtgtgtggtattttt 7020 tctttttaatgatttagtaagttaagttagtaagtttggatcttagatttaacatctaat 7080 atctttctaagtcagcaaatttggggagtgtgattcttggcatttcaaaaggatttttgt 7140 tttctttatgacagtatttatcaagagttcttttacataatacatcttaataaattttaa 7200 gtatagatttatgggtaaaataactcgcttaagtaaaataattcacactgtactttaaag 7260 ccttctgtatgtgtgcactatgctcaatatggtgatatgaacctaagttccagtctttac 7320 taagtagaaaatatgggcaaagtattaaaccccataatgccataataaaggtacagagtg 7380 ctgttctaggcaagacataatcatcatgctcctttctgtcacctaaacaagaaagcaagg 7440 atccacttaaacttttctctgaatttctagttagtgtctgtggaatttcataaactgaga 7500 gttcaggaggagaaatcttacattgtactgagaaatgaacccttaatgcctttctccttt 7560 aaaatgttttgactagttcagcctttgatattttatcacattgctttgaggcattgtaga 7620 ctgttttaatgcttgagaatttctgaaactcctaataatatcacacttcaaataatttca 7680 ggagatccatctccttaacttgtaaaccataacagtactggaaatttagaagtatgagaa 7740 aagaagaaaataaattattcaagcctatttatcttacacatcttttatcaactatgtata 7800 gggctttccagtcttttttcatgtttatggttttaatcattatatgtcctgtttactttt 7860 cactccaaacattgtatcatgcttttctttttttctttcttttttttttttttttttggc 7920 attcctacctcatgtgtttaaaaaagaaaaaaatgcaaactgaggcttggaaagactgga 7980 ctgatcacctagctattatacatgtgaattcctacaaatgaaaatgcccgaatactagaa 8040 aacgtggtactgtgtaaatagtattgattcaagtattagggtgatgacacagatcagatt 8100 gtgggaataaaatattgtacagctgtacagagaaagattttgttcatgagtttcagcaat 8160 gcaaagtacatatttagttataaattctaaataaatagtcgatttttgtttacctgtcat 8220 tttattatttcaatatatacttttcttcttttgataaaattcctgaattaaaaatatatc 8280 catatctaaggcagagttatgatcatagcagttacttgtaataatttattaatggtgttc 8340 aagtatagatggcacccatatctcctgctattgatttttttaagtaaaactttaaatgac 8400 aggtttatattttaatgtatgccttaagcatcttaattatcttatcagagttaaggcatt 8460 ttgagaatatttttgcaataccagccatatattcttcattttcttccactaaaatatcat 8520 cagattcacttttttttttaaactgtactttgtttttaacttcttggttgaggtcagcag 8580 catgctgcatttatataatagtaatattaaaattgtaaatttttttttaaagttacactt 8640 caaaaattaaaggggaaaaagtatttcaagtcagagggtttatcacaaagtttttctagg 8700 tctgttttatagcagtgaaacaggtatgccaagttgttaataaatgggcatgcattgctc 8760 ttaaattcttaaaattaaacttgatcagttgtgagggacagcttatgtctatgcattgcc 8820 taaaaattgtttctgtttgttggctttacttatagaaaatcttgatttttttttcattac 8880 ttgatatttttattctttgtttttcctgataagctgccaaatggtgtcacttaccacact 8940 ggaacatatcaagaccggttaacaaagctacaggataatcttcgccaactttcagttctc 9000 ttcaggaagctgagattggtatatgacaaatgcaatgaaaactgtggtgggatggatccc 9060 attccagtcgaggtaattttttgtgatagagggaggatgaatataaggtgtgaactagtg9120 tcaagacagttgttaatctttttcctctctctcctgttttagcaatatttccaggtttcg9180 aattttattggttttacttttgaccagagaagatgaaaatgtgatatatatgcataaaat9240 actactatatttttacattaacaatttcttgtagactttgacaggcttatagtaattttt9300 gttgttgttaggaagaaatgttttcctatggcaatttttattgctgtcagtgtctccttt9360 taattaagtcagggatatttttagtaggaattttaatagtatcatatacttttaaggtgg9420 ttttcatgaagtactttacagataaaatattatttttattattattaatttggattttat9480 atttagcccagtggaatcactatattttaaaaatatattctctaaatttaatcttgggtc9540 atacctatttagttaattaggcatatatttctataagtcaccctttcctgttgacaaatt9600 atatctctgccaccaaaggaagctggtaagagatgaccagagaaaaattaaactgcaaaa9660 atgcctaaaaagaaggggtcagagatgtgggagaaagatcagaaatgtggcctacatcgt9720 cagatgctacagagaagtcagggaaaataaggaatgaaaactgtccagtagattttgcta9780 gatgaaaattgttggtatatttgagtagagcttcagtgaaatgatagcagcgtgaagtct9840 gatcctagtcgggtgaagattaaacaggaaataagaagctggagccggcaagtggatatc9900 agtctttcaagaagctttgaaaaaaataagagaggaaagcagtagatggaaggagagatg9960 gaatcgaaggtttgcaggaggaagaggagcttccctccaaatagaagagataggaacatg10020 tttgaataataaaggaagaaagctgtagggagaggttgagagaaaggtgtataaataatg10080 aagacaggagggcagagtaatcagggcacaggaatggagactaatcgtcttcaggaggaa10140 ggacagctctgcattgagacagatgaaagaaagaaaaggagaatgtagacatgggtgagt10200 ttatatatatcccaagaatttgaagaagttttatcaggtgaatcctagttttaccagtca10260 tttacctttagcctattcttgcattggtaatacacgatggtaaaggctaggtgattcctg10320 aagtctgccacacttatgcggtaaattccatcactaaccatgcatacttaattacgattc10380 tgctagaagtttcattgctatataaatttgtttctcctacaaagaaaaagttctcacaac10440 tgcgaaaacttaagacatctagaagtttgcatagactctgaagttttagattgcaagtta10500 atgacaattatataatacagatccgtgaagttcttttctttttattgcagatgaaggaaa10560 gcctgttttatctatctcaatagactacaaatctcttaggggcaggaatcacatcttgtt10620 tggcattacttttatagtgtttcataattaaatatttgctgattgagtagggttttttta10680 aatttttattttattttattttattttttacttctcccatcttcctgctggaagtattgt10740 ttagttgtacatgacaaccaatgttataaaggctgttttgcaaaagaaacctgaaagggt10800 aatgcatagaatgcattgaaatggtcaactattaaagctcttatttcaggccatcacctg10860 aattgaaattatttttatacaaataaaagtataaaaattgaccttaatctgatatattct10920 ctatacaagtgattagaaaacttgaattatatttttgccttttaatttttgttaatcatt10980 cccagcaacttattccatatgtggaagaagatggctcaaagaatgatgatcgggctggcc11040 cacctcgttttgctagtgaagagaggcgagaaattgctgaagtaaataaagtgagttgtt11100 agtttttacattttatgttttagagttattgataaaaattactagggagtctgatgtaac11160 tccaaaacataatttgaattatcaaattacatgaagttgtttctgcggcaaacttcacct11220 atttagaatacagccaagaaatgaaaggtaagcagaaatgctgtctgaatagtcagaaga11280 gaaattgcaaaattcattcgataaagctcttagaagagctccttagacacttagtcacaa11340 tccatttacttttttcttttctgggcagaattctacctttctagcttatagcatatttgg11400 agcagcaaatgataaaaatttacaaatatgaaatgtgaatgtctcaacacctgaataaaa11460 aatttagagaactatcccagcattattatctactccaaccccacaaaaagcttgtaactg11520 gaatgtatttatcgctgaatactttcaaaagttcagaagataatgttcaaatagttctcg11580 tgaaacagagagaaatagggaaaggtgtcagttcattctggggatggaaatgtggcccca11640 gcttagcaaaacctaccagagatagcacagtgaataaaactttcagcatcactctttcac11700 tgacaaatatagattgccccttcctgaaattcttaattctaattaatagaactcatcagt11760 gtattaataatccaccactgccaaagagagtcttctgaggtatatgaagattttatagtt11820 cagtttgtatgtataatacatgatgtcagtaaactaccttgaaaaaagattttctcacat11880 gtctaaaagacatctgataaaattcaatactgttgctgattcaaaggacacttaaaacta11940 ttagcaagacagtgtgttttttttttaaatctttttcttttagataaatacccaacattt12000 ttcataaggtgaaatatagaaatatccttattaaaaacaagcatgaaataagatacttta12060 taacactatgtttaatattattttagaagttatagccagtacaaaaagacagtggacaaa12120 tgacagttgggaaacaggagttactatgtatgagcttgttagaatggtcaaagaaactca12180 actgatgaacaactagtctataagagttccgtaaaagggtctaagacaagactgatacca12240 tgctcttacaatgctgtaaacctattagattataagattctattgactcttgtaacaatt12300 aatattaagtagataggaattatgtcaataatgtgtatgaataaacttattaagatacat12360 aaaggaagctttgaatgattaaaagaacatataaagttcttggatgagcaatacatatta12420 taaaaataccagtcttgatttaatactcacagttttaatacagtaacaatccaaatccta12480 ataagaaagcatgcaaaaacaataggattttagtgcaataccaatcaataaacaaatcca12540 aataaaacccaaatggaatttttttcttgaaagtatacaaaaatatcaaagaaaatgttg12600 ataaatacgactaataaagcagaattcttagcttcaaaaattaaactgtaatatgaagca12660 tattataaaaatggtaattaggtgtataatactggcacaagaataggcagattagtaaaa12720 taatctagccagaaaaacagccttcttattgtacatgagaatttaatatgacagaagagg12780 gcttgtgtggaaaattagtctttagaaatagaaactttgggggcgaaattaagctagctt12840 ttacattacaccataaatcaaaatgaatttcagagttgaaaataagacctgcaaaacaaa12900 atattggataattttttaagtattttaaaactcggaagaaaggaaaaagatggatagatt12960 ttataaaataaaattttgtaacttttttaaaaggtgaaacacacatttagaaaactattt13020 ccaaaaaatctgattgtaatacatgacaatcataataaaaaactcacagtaaaaaattgg13080 tagagggtgaggaggagaatgggaacttttagtttagatcttgaaaaataactggtgttt13140 ggtagtctagatgtatgagacattcacttattaagacgtgttatatgtttctgttgataa13200 atatgtattacctcaataaatgtgttctgcttatgtttctctaatgatttttttataaaa13260 tataatgaaatatgtatataaataaaacaccattataataataactttggtttcagagta13320 ccacatgtgtaactaatgtaaacatagtttgttctgtaccatagaggattttataatatt13380 ttgtaaaaaataaagctgtgtaaattcagaaagatactttttctatatttacatttctct13440 tcaattttcattaagtttctcttttttcccaatttgataactcagaaataacatgtcttt13500 gacttttaaaagttctcttttataatcaaaatatggtaattctttggcagcatgtaacca13560 aaaaaaaaaatcttactatctactgacatagagttttgtggattttaagtaaaggatata13620 attgaaacaaggatatgatgtataaaaggacttctttctccccatgaggatttagctccc13680 tacagcctctctctcctggacccacctacataattgtttccctcaaacctttcagtgcac13740 ttatatcatatttggggttcagtaaataatctgtatcatataattatggcttcataagta13800 ttgtttattgctaagccgcatagtatactagggtcatatttccttgattgaataattttt13860 tgtttttctgaagttaattcttacctctttaattttctaaaattttagttagttttttat13920 gcatctatgacaacatctcagtactattttccaattttccacagtcagaatcatcagaca13980 gtttcatggttctcatcccaccctcccaagttttctatggtcttataggtctggatggaa14040 tgctggctaggcctgcttttcagctctggggctttccttcacctcttctgttggtttcac14100 tatttcttcagctcatgtcttcccttttcttggttttcttccttgttggtgatgtgcata14160 catcagtgacttctttagaaggaatgcagacacttaaattttgagtccttacatatttgt14220 ccagttgatagtttggctggctataaaattctatgttgtaagttatgtttcctcagaaat 14280 ttgaaggcattccataatctagtatagggtttggcagactttttgtttaaactactagat 14340 agtcaatcttttaaccattcctccagttttcattcccagggctcacctggtgcctctgag 14400 atggtctctgtctgtggttctccatggtggatcaggcgtattctcggtggcatccctctt 14460 gcatgcccaggccttctctgagtcactgaccactccctctttacttttcatctgaaaatt 14520 taccttgcatctgctgttgtgtcctttctgatcacttttctctgcatcagtttatagctt 14580 tttttattcctctcctgccacacactcaatagatacacaatgtttctgaaagtttgtgta 14640 tatttacggaatgaaagtattttaaatttttttccccatttttacttctgacattgagat 14700 ctcttcctttgattaaatgacttggatctgttctaagtgcttttaacttcaggataaaac 14760 atgttttagttaacgtgatatcaaactgatgggttattacaaagagaaaagaatcagttt 14820 aggtgttttaaatgaaccaggtttaaagctaaactctatttctgtgggaattgcttttta 14880 aaagacaatttagaggtaaagtaccttcttgagtatcatggggaagaactcatttgatga 14940 cctcataactctctttgtatgaattcttctacctatattttgagaaagcactgaaagatt 15000 aggtggtgggcacagggaaaagatgggagagggctaacatttggttaacttaaatttaaa 15060 tcccaaataaggaagaagaaaataaaattaactgctaaaaggcaaacaaattgttttttt 15120 tttttttttttaccaaaagggctttttaattcattccttaaaaaaatatataaatgtgaa 15180 ttaaatgtagcatattctgttactagaataatcttacctacagttaaccctagctactga 15240 catagagttttgtggattttaagtaaaggatataattgatacaaggatatgatgtataaa 15300 aggactttctccatatgaggatttagctctctacagcctctctctcctggacccacctac 15360 ataattgtttccctcaaacaattttattttattattttcctcaacttctttaagctctgt 15420 gtctccattctagaatataagattaaaatatatgattaaaagttttaatttttaaaatac 15480 tgagctcgtgatccattgatactttttttttttttttttttttgagagacaaaatctctc 15540 tctgtcacccaggctggagtgcagtggcacgatctcggctcactgggttcaagcaattct 15600 cctgcctcagtctcctgagtagctggggttacaggcgtgcaccaccatgcccggctaatt 15660 tttttgtgtttttagtagagatggggtttcaccatgttggtcaggctggtctcaaactcc 15720 tgacgtcgtgatcacctgcctcggcctcccaaagtgctgggattacgggcgtgagctact 15780 gcacccagccgatacatctttttttttttccgtaatggcacacacacccgagggtgtttc 15840 tcgtagtgaaaactggctatagatgataatgtactgatttgattatttaatcacatagat 15900 gctattaaaaataaagttcactgatttgatttgaccactatttataaatagctacattca 15960 taaacttaagtttttgtctctgtcaattatgttaatccacattcttatgtaaatataggt 16020 ttgctttttttttagttcatgcatttactaggtgatagagttttaaagttcacggaatct 16080 tagctggtaacactaaaacattgaaaatacatcttacctattatggtatggagtataaaa 16140 tgcatagtttttaataatggccattattaaatgtcttttttttttatactgaggagttat 16200 ttaaacaaaaataagttttgaaatgattggcaataatttttgtttgcttgcttatttttt 16260 ttccattcttgtaggttttgggagaagctttttgatttggcttttgttattttttcttca 16320 aacatgaaaatatttaaatatttgtgtgcctgctctctgacaggcattgtcctcagtacc 16380 agaatttcttccatgaagcagctggatgtgttgtcggctatatactcatttcacatgagt 16440 ggacaagtttcttcttggagaacataagactccagtggaaacaaagcagcacctgtttca 16500 tgtacccatttcatgttttgatgaatatttacagaaacaaaagtattgtgccatattttt 16560 aatacattggcatttggttgtattaaataagttgctttatagatctgtgtaatttataat 16620 aaaattatattatttgcattcaattaaaaagtcagaattttattacaatgaatattttct 16680 ttctttctaaataagctacctaaaataagtagatttatatacccctcagctgaccctcag 16740 tcattttcaagatattatcatatcattgtgtacttgaaattgtgcagaagaacatattga 16800 tggtgtgtttactatttagtcgttatggtctgtctggtttctaacaatacaaaaaccacc16860 aataactatcctggtgctatgaaactttgaggcacatgatttgtagtcagtatttgatgt16920 aaatcatcataagacaatatggctggttttaatagtcaaattcgtggtttatttaaatac16980 ttttcattttctcttaatatatcctggaatccttaagccaaatacccatttaaaggatgg17040 acacttgtttgagaccatatgctttaggtgcaggggtgtccaatcttttggcttccctgg17100 gccacattgtaagaagaagaattgttttgggccacatataaactatactaacattaatga17160 tagctgagaagccaaaatataaaaaaatcacgaaaaacatctcataatgttttaagaaag17220 cttacaaatttgtattgggctgcactcacagccgactggggccacattgcctgtgggcca17280 cagttggacaagcttgctttggggacatcagaatgagttttattttgcttaacgctcttt17340 tcttgatggtaaaatgaaattgaaaaagatagtaggtagtatttacaaggttggcattga17400 gtccatgcatttagaaagtaaaccattttttgtcaagtgtcacagcttttgtgtgaatat17460 tacattacatggtatcagttagcatcaactttttttaagtatttagtctacttacacatt17520 cgcctgttaatgtacaaaattgggcacagctgatggattttgaaaatagcagctgtcaac17580 gtctgttaaaatcaatcagtggcaagactgtgtatattaaatcctatatcgtgtcatgaa17640 aatttatttacactttagcctctaataagcataagtgtcagctttgctgttgatcatttt17700 catgacatgttttgactctggatattttatttgtatgctaatttttttaatttaaaactt17760 tgtggcatctttgagaaacgctatcccaagagccttataatatattaattatttaaaaat17820 tttctattccccagtgttgaatattatcttgaaaataccctacaaattttagttgttact17880 tttttaaaaaacaattacgaggcttaaataaaaaagagtaccagaatgtatatttccaaa17940 tgaggattcttgagagtagtcttattggtaggttatgcatttatttcagtggtgagacca18000 ttgtgatagccagctgcaagccctgagttgatactgattataaagtctgtttttaaccac18060 ccagatgaccaattgtcatcttatgtatgggtatgcagaaagtacaactggcagagacct18120 tagattttataatccaattacataaatgagaaaaccaagaagaaggaatttatctaagat18180 caaataccaagttagaggcaaagttggaactgggaccgagattgccagcctcccaatata18240 gcacttttatattcctctgttttgctgctactcttgattgctggcagaagtgttggtttc18300 ttggtctctgtgatgaaaaagactttataggcttggttattcttcatctttcccagcaat18360 ttgaatcaattttgttagatatatacaaccacttaaataacagactatcacttacttatg18420 ataacagaatatagaagatgggtcatctgaaaacttgtgcaagggagactaactaaaaat18480 accagcagtttttgtttcccgaagactgtgttaaagtgagctttagagtcgagccctgtg18540 tactgtatagtcctgtttttctctgactgcattgtgctagttctcttttcaaatgtgaat18600 taaattcagttatgaaagattattaaagtctctgtaaattgattttctattttctaataa18660 gagtttcttggtctttccttttagccacctatttcttgttacttccctaactctgaatag18720 aagcaatacaagcaacttcattttagtttgttttttaatgaaatcaaaagtaagattgtt18780 aggcctctttttaataagcatacaccaatttatctggaggtagtttaatatagtgtctta18840 aaattaattaagtatattatctccaaatgtaatgtttccaaggggttattgaataataat18900 aattattgttattataactagtaaacactctgaccagaagttctctatttctatcttgtt18960 ttatctaagcagacatttaccacataggatttgaagaaagaatggttaaaaagaagtcta19020 caaatcttagtcaagtagaatccaagtgtagagatacagagacacatgccgtgcacattt19080 caggcattcagcaaatcttagtagagaagattgcattgggtactctgccatttgtcttca19140 gtttttagaattagaaggagattgcaattcatgctataggcaaacagtgatttctgccgc19200 acttttgaaagactgattgcatgaggcattgtacttttattatattttctatatcgacag19260 tttacattttgtgcctaacagtaacatttgttgaatgtttacttatgtgtcaggcacaat19320 tctaatacttgacatgaatcagaccatttaatcttcataacagctcttatgaggtagttt19380 caattattatttccatttttctgatgtggaaactaaagtaccgggaggttaaataattag 19440 gtcagtgttgtaagtttcatagtgaaggatctggccgggtgtgatggctcatgcctgtaa 19500 tcccagcactttgggaggccaagacgggcagatcacttgaagtcaggagaccagcctggc 19560 caacatggcaaaacctcctctctactaaaaatacaaaaaaactagatgggtgtggtggct 19620 ggagcctgtaattctagctactcaggaggctgaagcaggagaatcgctgaaacccaggag 19680 gctgagattgcagtgagccaagattgcgccactgcactgcagcctgggaaaaaaaaaaaa 19740 aattcgtagtgaaggatctgaatgacttccagagatctggctctagagtatgggctcttg 19800 accgttaggttatgctgccccttagaaaacttatatgcttatgttttataacaaataaat 19860 gtttagctgatttattgatttgtttatggtggaagtggaagagtaagcagggcctggggc 19920 cggggctggggccatagacctctgcaggctggatttttagaattcaggatccctgtcagc 19980 aaatatttaaggccagaagagggttgagaccatctgccttatagggttatttagcttaat 20040 ttctgtctgtatcattataatcctaacttatgatattttaatcttctaacttcatggtct 20100 atatttcctgaaacatttttgataaattcttttgtttctacagaaactcaaacagaagaa 20160 tcaacagctgaaacaaattatggatcaattacgaaatctcatctgggatataaatgccat 20220 gttggcaatgaggaactaagctgatatttaaatttcctgctttacacatgttataccatt 20280 gttttttccctcaagtattttttccctgtgaagaagattatttatctgcttttattttag 20340 tcactaaaactaaagtttttatttttacattgtgatttttacattaaaatattaactttt 20400 ttaatgctattttatgaaagattattgtaataaactttgatggggtttgtattttggtta 20460 atcttcatgaattgaataattgtttttttaaagcaaaataaagttttttaaataaatgtt 20520 aatatttgattaatggatttactcattccaattacccttttagaagaaaaatatttaaat 20580 atttctgcagataaaagt 20598

Claims (17)

What is claimed is:

1. An isolated polypeptide comprising at least nine contiguous amino acid residues of SEQ ID NO:2 or SEQ IDNO:4.

2. The isolated polypeptide of claim 1 having from 15 to 1500 amino acid residues.

3. The isolated polypeptide of claim 2, wherein said at least nine contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4 are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ID NO:7.

4. The isolated polypeptide of claim 1 comprising at least 30 contiguous residues of SEQ Id NO:2 or SEQ Id NO:4.

5. The isolated polypeptide of claim 1 comprising:
residues 41-55 of SEQ ID NO:2 residues 56-77 of SEQ ID NO:2;
residues 78-92 of SEQ ID NO:2;
residues 78-92 of SEQ ID NO:4;
residues 93-110 of SEQ ID NO:2;
residues 111-125 of SEQ ID NO:2;
residues 111-125 of SEQ ID NO:4;
residues 126-148 of SEQ ID NO:2;
residues 126-148 of SEQ ID NO:4; or residues 149-163 of SEQ ID NO:2.

6. The isolated polypeptide of claim 1 comprising:
residues 41-163 of SEQ ID NO:5;
residues 34-163 of SEQ ID NO:5;
residues 34-178 of SEQ ID NO:5; or residues 18-178 of SEQ ID NO:5.

7. The isolated polypeptide of claim 1 comprising:
residues 41-163 of SEQ 1D N0:2 or SEQ ~ N0:4;
residues 34-163 of SEQ m N0:2 or SEQ >D N0:4;
residues 34-178 of SEQ 1D N0:2 or SEQ ~ N0:4; or residues 18-178 of SEQ m N0:2 or SEQ m N0:4.

8. An expression vector comprising the following operably linked elements:
a transcription promoter;
a DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of:
residues 41-55 of SEQ m N0:2 residues 56-77 of SEQ ~ N0:2;
residues 78-92 of SEQ 1D N0:2;
residues 78-92 of SEQ m N0:4;
residues 93-110 of SEQ 1D N0:2;
residues 111-125 of SEQ 1D N0:2;
residues 111-125 of SEQ m N0:4;
residues 126-148 of SEQ 1D N0:2;
residues 126-148 of SEQ m N0:4; and residues 149-163 of SEQ )D N0:2; and a transcription terminator.

9. The expression vector of claim 8 wherein the DNA segment comprises nucleotides 52 to 534 of SEQ m N0:6.

10. The expression vector of claim 8 wherein the polypeptide comprises:
residues 41-163 of SEQ m N0:2 or SEQ ID N0:4;
residues 34-163 of SEQ 1D N0:2 or SEQ m N0:4;
residues 34-178 of SEQ m N0:2 or SEQ m N0:4; or residues 18-178 of SEQ ~ N0:2 or SEQ m N0:4.

11. An expression vector comprising the following operably linked elements:
a transcription promoter;

a DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of::
residues 41-163 of SEQ ID NO:5;
residues 34-163 of SEQ ID NO:5;
residues 34-178 of SEQ ID NO:5; or residues 18-178 of SEQ ID NO:5; and a transcription terminator.

12. The expression vector of any of claims 8-11 further comprising a secretory signal sequence operably linked to the DNA segment.

13. A cultured cell into which has been introduced the expression vector of any of claims 8-12, wherein said cell expresses said DNA segment.

14. A method of making a polypeptide comprising:
culturing the cell of claim 13 under conditions whereby the DNA segment is expressed and the polypeptide is produced; and recovering the polypeptide.

15. A polypeptide produced by the method of claim 14.

16. An antibody that specifically binds to the polypeptide of claim 15.

17. A method of detecting, in a test sample, the presence of an antagonist of zalpha33 activity, comprising:
culturing a cell that is responsive to zalpha33;
exposing the cell to a zalpha33 polypeptide in the presence and absence of a test sample;
comparing levels of response to the zalpha33 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison the presence of an antagonist of zalpha33 activity in the test sample.